Hematology and coagulation disorders

Mechanisms of hemostasis

Hemostasis is a complex physiologic process that regulates the formation and dissolution of a clot. Several cells and proteolytic enzymes are critical to this process, including platelets, thrombin, and plasmin. All of their actions are tightly regulated so that effective clotting occurs only at the site of vascular injury while maintaining blood flow in other parts of the circulation. The three phases of hemostasis are the formation of a platelet plug, coagulation, and fibrinolysis, also called primary, secondary, and tertiary hemostasis.

Primary hemostasis

The formation of a platelet plug is essential to hemostasis ( Fig. 49.1 ). After vascular injury, platelets bind directly to collagen exposed in the subendothelium via two major receptors: integrin α2β1 and glycoprotein (GP) VI. This initial binding of the platelet to subendothelial collagen is referred to as platelet adhesion. Under turbulent flow conditions, von Willebrand factor (vWF) undergoes a shear-mediated conformational change, thus allowing it to bind to the GP Ib platelet receptor ( ). The adhesive interaction between vWF and the platelet GP Ib receptor effectively tethers the platelet, thus allowing stronger bonds to be formed between collagen and the α2β1 integrin and the GP VI receptors. The importance of the vWF-GP Ib interaction is illustrated by the pathologic conditions that occur in the absence of either the ligand or receptor such as von Willebrand disease (vWD) and Bernard-Soulier syndrome, respectively.

The binding of platelets to subendothelial collagen triggers platelet activation. During this process, substances such as thromboxane A ₂ (TXA ₂ ) and adenosine diphosphate (ADP) are released from the platelet α- and dense-granules. These substances strengthen interactions between adherent platelets and promote the recruitment of additional circulating platelets into the growing platelet plug. Also, new negatively charged phospholipids become expressed on the platelet surface membrane. These negatively charged phospholipids facilitate the adhesion of various coagulation factors to the activated platelet surface. Finally, the platelet glycoprotein IIb-IIIa receptor is converted from an inactive to an active form that is capable of binding both fibrinogen and vWF to promote aggregation. This localized process tightly controls the formation of a platelet plug so that it is limited only to areas of vascular injury. Propagation of the platelet plug beyond the site of injury is also restricted by powerful platelet aggregation inhibitors and vasodilators produced by intact noninjured endothelial cells ( ).

Secondary hemostasis

Platelet aggregation occurs in conjunction with the activation of coagulation factors (secondary hemostasis) on the platelet surface in order to support thrombin generation and the formation of a fibrin clot. Our understanding of secondary hemostasis has evolved over time—from the traditional “cascade model” consisting of independent plasma-based intrinsic and extrinsic coagulation pathways into a “cell-based” concept in which coagulation factors interact on the surfaces of tissue factor (TF)–bearing cells and activated platelets at the site of vascular injury ( Fig. 49.2 ) ( ).

According to the cell-based model ( ), TF is the primary physiologic initiator of coagulation. Upon injury, TF in the vascular subendothelium becomes exposed to circulating blood. Factor (F) VII has an extremely high affinity for TF. Once exposed, FVII rapidly binds TF and is converted to its activated form, activated FVII (FVIIa). The newly formed FVIIa/TF complex catalyzes the activation of FX to FXa. This sequence of activations was formerly called the “extrinsic pathway” of coagulation. The FXa on the surface of TF-bearing cells generates a small amount of thrombin (IIa) from prothrombin (II). This initial small amount of thrombin, although insufficient to initiate fibrin formation, activates platelets and factors V, VIII, and XI. Thus activated platelets participate in primary hemostasis and provide a negatively charged surface upon which further coagulation processes can occur ( ).

The FVIIa/TF complex also activates FIX into FIXa. The activation of FIX was formerly part of the “intrinsic pathway.” In this pathway, the contact activation factors (FXII, FXI, prekallikrein [PK], and high-molecular-weight kininogen [HMWK]) are activated as blood comes into contact with negatively charged substances such as kaolin. On the activated platelet surface, FIXa joins with FVIIIa to form the “tenase” complex (FVIIIa/FIXa). This complex activates large amounts of FXa, which may then join with its cofactor, FVa, to form the “prothrombinase” complex (FXa/FVa). The “prothrombinase” complex subsequently generates a large-scale conversion of prothrombin into thrombin.

As a major regulator of hemostasis, thrombin plays several roles. Its primary function is to mediate the proteolytic cleavage of fibrinogen into fibrin monomers. Fibrin monomers then polymerize into an insoluble fibrin network, which forms the essential infrastructure of the clot. In addition, thrombin amplifies the activation of other coagulation factors (including itself) and platelets, initiates the process of fibrinolysis, and stimulates the vascular endothelium to release vasoactive substances and inflammatory mediators.

Similar to the platelet aggregation, coagulation must be limited to avoid thrombotic occlusion in adjacent normal areas of the vasculature. Endothelial cells and circulating protease inhibitors play a major role in confining coagulation to the site of injury. When thrombin diffuses downstream from the site of injury and reaches intact endothelial cells, it binds to a protein on the endothelial cell surface called thrombomodulin (TM). The thrombin/TM complex activates protein C, which then binds to its cofactor, protein S, to inactivate FVa and FVIIIa. In addition, circulating protease inhibitors, namely antithrombin (AT) and tissue factor pathway inhibitor (TFPI), are present near the endothelial cell surface, where they can inhibit thrombin and TF, respectively. Protease inhibitors also impose a threshold effect on the coagulation process ( ). Coagulation will not proceed unless procoagulant factors are activated in sufficient amounts to overcome these circulating inhibitors.

Tertiary hemostasis

The final phase of hemostasis, termed fibrinolysis, is critical to maintaining and regulating patency of the vascular system ( Fig. 49.3 ). Plasmin is the principle enzyme of the fibrinolytic system and acts to lyse mature fibrin deposits and thrombi within the vessel into degradation products, the smallest of which are known as d-dimers. Plasmin is activated from its zymogen form, plasminogen, by the enzymatic action of tissue plasminogen activator (tPA). tPA only activates plasminogen that is bound to fibrin, thus limiting fibrinolysis to the site of clot formation. The main inhibitor of this process is plasminogen activator inhibitor-1 (PAI-1). Therefore the degree of clot lysis depends on the balance between tPA and PAI-1 activity ( ).

Standard coagulation tests

Citrated plasma has been used for decades to assess the initiation of clot formation in routine coagulation testing. This is accomplished relatively easily in hospital laboratories by photometric tests such as the activated partial thromboplastin time (aPTT), prothrombin time (PT), and international normalized ratio (INR). Although the aPTT, PT, and INR are appropriate to monitor anticoagulation, they were not originally designed as screening tests. Studies indicate that these tests have little value in predicting perioperative bleeding and rarely lead to a change in the clinical management of a patient ( ). Nevertheless, there are occasional reports in which routine preoperative coagulation testing identifies individuals with bleeding disorders that would have otherwise gone unrecognized ( ).

The aPTT is used to assess suspected deficiencies in procoagulant proteins of the intrinsic and common pathways, including factors VIII, IX, X, XI, XII, and II and fibrinogen. In the normal population, there is a large degree of interindividual variability in response to the aPTT, which is reflected in its broad reference range. Most commonly the aPTT is used to guide and monitor anticoagulation in patients requiring therapeutic levels of intravenous heparin. The PT/INR is used to assess suspected deficiencies in procoagulant proteins of the extrinsic and common pathways, including factors V, VII, X, and II and fibrinogen. In the early 1980s the World Health Organization introduced the INR in order to overcome the variability seen in PT values that resulted from differing analytical systems used to perform the test. Because different manufacturers produce different thromboplastin reagents, the INR was devised to normalize the test results against an international standard ( ). The PT/INR is most commonly used to guide and monitor anticoagulation in patients requiring therapeutic levels of coumadin or other vitamin K antagonist drugs. Table 49.3 summarizes several conditions that may be present based on abnormal aPTT and PT testing.

Viscoelastic coagulation tests

Given our increased understanding of the contribution of platelets, leukocytes, and phospholipid-bearing cells to the process of coagulation, interest has shifted from the use of plasma-based tests to the use of whole blood for coagulation monitoring ( ). Point-of-care monitoring devices capable of assessing the viscoelastic properties of whole-blood coagulation during surgery include TEG (Hemoscope Corp., Niles, IL), rotation thromboelastometry (ROTEM; Pentapharm GmbH, Munich, Germany), and the Sonoclot Analyzer (Sienco, Inc., Arvada, CO). These tests analyze real-time clot development in a visual display. The use of celite, kaolin, or tissue factor to activate coagulation and speed the attainment of TEG and Sonoclot values has allowed these monitors to become real-time, point-of-care coagulation monitors in the operating room. The addition of protamine or heparinase to the sample can neutralize any potential circulating heparin, thus allowing clinicians to obtain a thromboelastogram in anticoagulated patients and even during cardiopulmonary bypass (CPB). Activated TEG tracings containing heparinase and performed during CPB at the time of rewarming produce results similar to activated tracings performed after protamine administration ( ). Compared with standard laboratory coagulation testing, these point-of-care devices provide information on the specific impaired hemostatic defect that allows for the timely institution of targeted treatment.

The TEG and ROTEM instruments produce similar graphic tracings of clot development ( Fig. 49.4 A). They differ, however, in the methodologies employed. These methodologies are explained in detail in an excellent review ( ). Briefly, TEG uses a cylindrical cup that oscillates through an angle of 4 degrees, 45′as it holds the blood sample. A pin, suspended by a torsion wire, is immersed in the rotating cup. The torque of the rotating cup is transmitted to the pin only after fibrin-platelet bonding has linked the cup and the pin together. Thus the magnitude of the pin motion is determined by the strength of the fibrin-platelet bonds of the forming clot. The rotation of the pin is converted by a mechanical-electrical transducer to an electrical signal, which is displayed as the typical TEG tracing. Conversely, the ROTEM instrument initiates the rotational motion in the pin, not the cup, and this motion is transmitted by an optical detector, not a torsion wire. Nomenclature and reference ranges also differ between the TEG and ROTEM instruments.

Measurable TEG values include the time until initial clot formation (reaction time or R), kinetics of fibrin formation (kinetics or K and α angle), maximal clot strength (maximal amplitude or MA), and clot lysis (CL). The coagulation index (CI) and shear modulus (G) may also be calculated (equations available through Thrombelastograph@ Operations Manual and CTEG User’s Guide, Haemoscope Corp.) and have been shown to be more sensitive measures of coagulation and actual clot strength than individual TEG parameters alone ( ). Similar values from the ROTEM device also provide information on the rapidity of clot formation (clotting time or CT), kinetics of fibrin polymerization (clot formation time or CFT), strength of the formed clot (maximal clot firmness or MCF), and presence of fibrinolysis (LY).

The function of the Sonoclot Analyzer has also been well described ( ). A normal trace is depicted in Fig. 49.4 B. This device provides information on the entire hemostatic process both as a qualitative graph, known as the Sonoclot signature, and as quantitative results: the activated clotting time (ACT), the clot rate (CR), and the platelet function (PF). To perform a Sonoclot analysis, a blood sample is placed in a cuvette containing different coagulation activators and inhibitors. After an automated mixing procedure, a plastic probe mounted on a transducer head is inserted into the sample and oscillates vertically within the sample. The changes in impedance imposed by the developing clot on the oscillating probe are measured and recorded over time. The ACT provides information on the initial phase of clot development, from the time of activation of the sample to the beginning of fibrin formation. Next, the CR is determined by the maximum slope of the Sonoclot signature graph and measures the kinetics of fibrin formation. Finally, platelet function is assessed by the quality and timing of clot retraction.

Although whole-blood coagulation tests may be most appropriate for evaluating the global integrity of the clotting process, several concerns exist regarding their use. First, the results of any given test are specific to the type of equipment and activators employed. Thus all modifications need to be considered when interpreting test results. Second, these instruments require that quality controls and routine maintenance checks be performed on a regular basis. Nonlaboratory personnel must be trained to accurately perform these tasks in order to ensure the accuracy of the devices. Third, evidence exists that patient age and potentially gender may significantly affect the results of these tests. Age-specific reference ranges have been published for both TEG and ROTEM values to aid in the interpretation of their results to the pediatric population ( ; ; ; ). Finally, one analytical study of the Sonoclot Analyzer suggested that the results of the platelet function assay may in fact be influenced by platelet count ( ).

Transfusion algorithms

Monitoring the coagulation system intraoperatively and using transfusion protocols result in less blood product utilization in both adult and pediatric patients. Most published guidelines recommend standard plasmatic coagulation tests to diagnose coagulopathy and guide hemostatic therapy, but these tests suffer from long turnaround times, insufficient insight into the complexity of acquired intraoperative coagulopathies, and a bias toward quantity and not quality of function. Viscoelastic point-of-care tests may be advantageous in providing evidence-based, individualized treatment strategies. In adults, algorithms based on point-of-care viscoelastic devices have been established to guide the management of intraoperative bleeding and have resulted in a decrease in blood product usage compared with therapeutic interventions based on standard coagulation values or clinical judgments of the appearance of the operative field ( ; ). In pediatric cardiac surgery and craniosynostosis, algorithms using viscoelastic tests have been associated with reduced blood product transfusions and potential cost savings ( ; ; ; ). Pediatric trauma and liver transplantation are other disciplines associated with high blood product utilization where transfusion algorithms may prove to be beneficial ( ; ).

Von willebrand disease

vWD is the most common inherited bleeding disorder, with an estimated prevalence of approximately 1 in 10,000 individuals ( ). First described in 1926 by the Finnish physician Erik von Willebrand, vWD is caused by quantitative or qualitative deficiencies of vWF, a glycoprotein important for maintaining normal hemostasis ( ). The most frequent presentation in children with vWD is easy bruising or epistaxis, although certain subtypes are associated with more severe bleeding.

Hemophilia

Hemophilia is an X-linked recessive bleeding disorder caused by a deficiency of functionally active FVIII (hemophilia A [HA] or “classic” hemophilia) or FIX (hemophilia B [HB] or Christmas disease) ( ; ). Over 2000 causative mutations have been identified in the FVIII gene and over 1000 have been found in the FIX gene ( ). Although most cases of hemophilia occur by genetic passage of these mutations, one-third of cases arise from de novo mutations ( ). HA comprises 80% to 85% of cases of hemophilia and occurs in approximately 1 in 5000 male births, and HB occurs in approximately 1 in 30,000 male births ( ). Hemophilia is significantly less common in females because both X chromosomes would have to carry a mutation but can be seen when both parents carry a mutation (father with hemophilia and mother a heterozygous carrier) or in females with Turner syndrome (XO) ( ; ).

Lack of functioning FVIII or FIX in individuals with hemophilia prevents formation of the FVIIIa/FIXa “tenase” complex and thus prevents activation of FX and subsequent thrombin generation (thus the bleeding propensity). Sites of bleeding vary with age. Newborns bleed from heel sticks or circumcisions or may develop a delivery-associated intracranial hemorrhage. Infants will bleed orally as teeth begin erupting and potentially into joints (usually ankles) as they begin crawling. Toddlers, older children, and adolescents will bleed into joints, soft tissues, and gastrointestinal and genitourinary tracts in addition to intracranially with associated trauma ( ; ).

Hemophilia can be classified by the amount of residual plasma factor level. The degree of associated bleeding worsens with lower residual factor levels, although the coinheritance of thrombophilic abnormalities (deficiencies of antithrombin, protein C, or protein S; factor V Leiden; prothrombin gene mutation G20210A; and hyperhomocysteinemia) may occur and will moderate the bleeding propensity at any given factor level. Mild cases have 5% to 40% of normal factor levels and bleed after provocation such as surgery, dental extractions, or accidents. Moderate cases have 1% to 5% of normal factor levels and bleed into joints or muscles after only minor injuries. Severe cases have <1% of normal factor levels and bleed spontaneously into joints and muscles. Bleeding into a joint results in inflammation and hypertrophy of the synovial lining, which promotes recurrent bleeding (“target joint”) and progressive joint damage usually involving the knees, ankles, and elbows. Development of hemophilic arthropathy significantly influences quality of life in hemophiliacs. The highest number of deaths, however, are caused by intracranial hemorrhages ( ; ; ).

The clinical manifestations of HA and HB are similar, but HB seems overall to be less clinically severe. Individuals with HB have fewer bleeding episodes, require less factor concentrate infusions, undergo fewer joint procedures, and suffer fewer physical limitations than those with HA. Less severe gene mutations and the facts that “normal” plasma FIX levels are usually 50 times those of FVIII and the half-life of FIX is twice that of FVIII (24 versus 12 hours) may help explain the phenotypic differences between HB and HA ( ; ; ; ).

Hemophilia is usually diagnosed because of a known family history or during evaluation of a bleeding event. Laboratory analysis will reveal an isolated prolongation of the aPTT in the face of a normal PT, thrombin time, platelet count, and bleeding time. The diagnosis is confirmed by FVIII or FIX assays. Because FIX levels are physiologically low in neonates, measurement should be repeated at 6 to 12 months of age in infants suspected of having HB to confirm the diagnosis. It should be remembered that FVIII levels are also low in types 2N and 3 vWD, so these conditions must be considered in individuals with low FVIII levels ( ; ; ).

Management of hemophilia strives to increase the plasma concentration of the deficient factor sufficiently to control traumatic bleeding, to provide hemostatic coverage for planned invasive procedures, and/or to prevent spontaneous bleeding episodes ( ). Raising factor FVIII or FIX levels to >30% is usually adequate to manage minor bleeding or minor surgery. Levels >50% are needed for more significant bleeding or more invasive procedures, and sustained levels of 100% will be necessary for life-threatening bleeding such as intracranial hemorrhage ( ; ). In cases of mild HA (where life-threatening and spontaneous bleeding are rare), administration of DDAVP usually accomplishes these goals, at least for the short term, by releasing vWF and FVIII from endogenous stores ( ; ). In all other cases, including all cases of HB, factor replacement is the mainstay of therapy ( ). Both plasma-derived and recombinant factor replacement products are available. In patients with severe hemophilia, continuous long-term prophylactic administration of factor concentrates is used to maintain factor trough levels >1% in an attempt to prevent spontaneous bleeding episodes, most frequently into joints. Prophylactic therapy is ideally begun as early as after the first joint bleed and has been shown to attenuate the development of chronic arthropathies and thus improve quality of life ( ; ; ).

Complications of therapy with factor concentrates include the transmission of transfusion-related infections and the development of inhibitors to the transfused factors. Plasma-derived factor concentrates are made from plasma pools of up to 15,000 to 20,000 donors. Whereas many transfusion-related viral infections occurred in past decades, current viral inactivation techniques and the use of nucleic acid tests (NATs) have essentially eliminated this concern. Nevertheless, recombinant products are now produced and are considered the treatment of choice because their use eliminates this risk. Currently, the development of neutralizing immunoglobulin G (IgG) inhibitor alloantibodies to FVIII or FIX is the most challenging treatment-related complication. These inhibitors render replacement therapy with either plasma-derived or recombinant products ineffective and develop in up to 30% of individuals with severe HA and in up to 5% of those with severe HB. The major determinant for inhibitor development is the presence of large gene mutations causing a complete lack of endogenous FVIII or FIX production, although multiple other factors, both genetic and treatment-related, play roles. Inhibitor levels are measured in Bethesda units (BU), where 1 BU is the amount of antibody that inactivates 50% of factor after 2 hours of incubation at 37° C. Management of bleeding in the presence of inhibitor alloantibodies depends on the antibody titer. In the presence of low titers (<5 BU), high doses of factor concentrates can be effective. With high titers (>5 BU), agents that “bypass” the FVIIIa/FIXa “tenase” complex are needed. Available agents include rFVIIa or FEIBA ( F actor E ight I nhibitor B ypassing A gent), an activated prothrombin complex concentrate (APCC) containing factors II, VII, IX, and X. APCC and rFVIIa can also be used as prophylactic agents to prevent spontaneous bleeding or to provide perioperative hemostatic coverage in hemophiliacs with inhibitors. “Bypassing” agents, however, are not a panacea. They do not normalize thrombin generation, the hemostatic response to them is less predictable than to factor concentrates, they are more costly and more difficult to monitor, and they carry a thrombotic potential. ( ; ; ; ; ; ; ; ; ; ).

The perioperative management of a patient with hemophilia should integrally involve the help of a knowledgeable hematologist. In addition to factor concentrates and “bypassing” agents, the use of antifibrinolytics, fibrin sealants, and local measures may be appropriate. Antiplatelet drugs (including aspirin and NSAIDs), intramuscular injections, and epidural or subarachnoid blocks should be avoided. Vascular access may be an issue in individuals who have received long-term prophylactic factor infusions. The future development of less immunogenic, longer-acting, and possibly even oral factor replacement products or of a definitive cure by gene therapy holds promise for individuals with hemophilia.

Inherited platelet disorders

Although most platelet disorders are acquired and result from drug effects or consequences of metabolic diseases, there is a diverse group of inherited platelet disorders (IPDs) that affect platelet production, morphology, and function ( ). These disorders are rare, and the genetic defects causing many of these disorders have yet to be conclusively identified ( ). Patients with these disorders usually present with mucocutaneous bleeding (easy bruising, gingival bleeding, and epistaxis) in addition to menorrhagia in female patients but rarely with spontaneous life-threatening bleeding. However, unexpected excessive bleeding after trauma, surgery, or labor and delivery can be encountered as an initial presentation in affected patients ( ; ). Multiple classification systems have been used to organize these defects, including pattern of inheritance, specific genetic defect, stage at which platelet production is disrupted, and platelet size ( ; ; ; ; ; ). However, none of these classifying schemes seems to include all of the known IPDs. This section will not attempt to describe the intricate details of platelet production or all of the heretofore identified IPDs. Instead, we will briefly focus on a few of the more recognized disorders.

Bernard-Soulier syndrome (BSS) and Glanzmann thrombasthenia (GT) are disorders of platelet surface receptors. Patients with BSS have defective expression or dysfunctional forms of the GP Ib-IX-V surface receptor. This receptor binds platelets to vWF on activated endothelial cells to promote coagulation and to thrombospondin on activated leukocytes to participate in the inflammatory cascade. Its cytoplasmic domain influences platelet size and shape and participates in activation of the GPIIb/IIIa platelet surface receptor. Thus deficiencies or defectiveness of the GPIb-IX-V receptor have significant consequences. BSS is characterized by abnormally large platelets, moderate to severe thrombocytopenia, and a bleeding diathesis resulting from the loss of vWF-dependent platelet adhesion to exposed subendothelium of damaged blood vessels. It is inherited as an autosomal recessive trait, except for its Bolzano variant, which is caused by an autosomal dominant functional defect in GPIb-IX-V receptors. The severity of the bleeding diathesis in patients with BSS is variable for ill-defined reasons. Platelet aggregation studies are pathognomonic in the diagnosis of BSS where normal aggregation is seen in response to ADP, collagen, epinephrine, and arachidonic acid but is absent in the face of ristocetin. Flow cytometry will show absent or severely reduced surface expression of the GPIb-IX-V receptor in patients with biallelic BSS and approximately 50% expression in patients with monoallelic BSS. Management of BSS includes adjunctive therapies such as antifibrinolytic agents, topical thrombin, DDAVP, or recombinant activated factor VII in situations of minor bleeding. Platelet transfusions may be required for severe bleeding or during surgical procedures but predispose to the development of alloantibodies against the GPIb-IX-V receptor, which may neutralize the effectiveness of future transfusions. Hematopoietic stem cell transplantation has been used successfully to cure BSS ( ; ; ; ; ; ; ).

GT is caused by deficiencies of or functional defects in platelet surface GPIIbIIIa receptors (α _IIb β ₃ integrin). These receptors bind fibrinogen to mediate cross-linking of adjacent platelets in a forming clot with resulting platelet aggregation and clot retraction. GT is an autosomal recessive trait characterized by normal-sized platelets and normal platelet counts. The associated bleeding diathesis is variable, is dependent on the type of mutation (two genes on chromosome 17 are involved), and can be severe. Platelet aggregation studies are again central to the diagnosis. GT is the only disorder where platelet aggregation is absent in the face of all agonists (ADP, collagen, epinephrine, arachidonic acid) but is preserved in response to high-dose ristocetin. Flow cytometry confirms the deficiency of GPIIbIIIa receptors and allows classification of GT into type I (<5% residual receptors), type II (10% to 20% residual receptors), and variant types (>20% receptors but dysfunctional). Treatment is with adjuvants and, when in crises, platelet transfusions. Stem cell transplantation may provide a cure ( ; ; ; ; ).

Gray platelet syndrome (GPS) is an autosomal recessive disorder characterized by absent or decreased α-granules in platelets. α-Granules contain proteins, including vWF, fibrinogen, thrombospondin, factor V, fibronectin, and platelet-derived growth factor. Patients with GPS demonstrate macrothrombocytopenia, a mild to moderate bleeding diathesis, and splenomegaly. These patients also frequently develop bone marrow fibrosis. Diagnosis is facilitated by a characteristic pale appearance of platelets on peripheral blood smear and by the reduction or absence of α-granules in platelets on electron microscopy. Treatment is symptomatic with the discretionary use of antifibrinolytics, platelet transfusions, and occasionally splenectomy ( ; ; ; ; ).

Hermansky-Pudlak syndrome (HPS) and Chediak-Higashi syndrome (CHS) are autosomal recessive disorders caused by deficiencies of dense granules in platelets. These granules contain nonprotein molecules such as ADP, adenosine triphosphate (ATP), calcium, magnesium, and serotonin. Patients with either of these disorders demonstrate a normal platelet count but abnormal serotonin-dependent platelet aggregation and moderate to severe propensities to bleed. HPS is characterized by oculocutaneous albinism and, in some subtypes, pulmonary fibrosis. CHS is associated with severe immune defects and progressive neurologic dysfunction. The hallmark of CHS is the presence of giant inclusion bodies in a variety of granule-containing cells, including platelets. Diagnosis is facilitated by the lack of dense granules in platelets on electron microscopy ( ; ; ).

Wiskott-Aldrich syndrome (WAS) is an X-linked disorder resulting from a mutation in the WAS gene with subsequent abnormalities in the production of WAS protein, an important regulator of cytoskeletal development in hematopoietic stem cells. WAS is characterized by moderate to severe thrombocytopenia and severe immune deficiency. Morbidity and mortality in WAS are associated not only with bleeding but also with infections. A mutation in the WAS gene that only partially impairs WAS protein function results in thrombocytopenia but no immune deficiency. This disorder is called X-linked thrombocytopenia (XLT) and has a relatively mild clinical course. WAS and XLT are the only IPDs that result in small platelet size. Poor platelet aggregation is found in these disorders. Flow cytometry to assess for WAS protein expression and gene sequencing are helpful in confirming these diagnoses. Prophylactic antibiotics and intravenous immunoglobulin (IVIG) infusions are administered to patients with WAS to protect against opportunistic infections. Hematopoietic stem cell transplantation is the treatment of choice and the only hope for cure for those with WAS ( ; ; ; ; ; ).

Immune thrombocytopenia

Immune thrombocytopenia (ITP), formerly called idiopathic thrombocytopenic purpura, is an immune-mediated acquired thrombocytopenia that has been described in both children and adults. ITP is defined by a platelet count of less than 100 × 10 ⁹ /L without concurrent leukopenia or anemia. ITP is caused by accelerated destruction of otherwise normal platelets augmented by reduced platelet production. Autoantibodies produced by B lymphocytes against platelet GP receptors, especially GPIIb/IIIa (fibrinogen receptor), GPIb/IX (vWF receptor), and GPIa/IIa (collagen receptor), coat platelets and promote their clearance by the reticuloendothelial system (mainly by the spleen) within a few hours of their entering the circulation (normal lifespan is 8 to 10 days). These autoantibodies also target the same receptors on megakaryocytes in the bone marrow, resulting in reduced platelet production. Additionally, cytotoxic T lymphocyte–dependent and complement-dependent mechanisms may be involved because antiplatelet autoantibodies are found in only 60% of patients with ITP ( ; ; ; ; ; ).

In 2009, an international working group of recognized experts in the field proposed standard definitions of this disease ( ). “Primary ITP” describes an isolated autoimmune thrombocytopenia in the absence of other etiologies that can potentially cause thrombocytopenia. “Secondary ITP” includes all forms of immune-mediated thrombocytopenia that are the result of an underlying disease or drug exposure. Diseases associated with secondary ITP include common childhood viral illnesses; immunizations (especially measles, mumps, rubella [MMR]); infections with Helicobacter pylori, cytomegalovirus, varicella-zoster virus, hepatitis C virus, or human immunodeficiency virus; malignancies; common variable immunodeficiency; and autoimmune disorders such as antiphospholipid syndrome and systemic lupus erythematosus. These disorders may result in the production of antibodies that cross-react with platelets, leading to their destruction, or may infect megakaryocytes, leading to impaired platelet production ( ; ). Distinction between primary and secondary ITP is important because of differences in treatments and natural histories of the processes.

The 2009 international working group also defined phases of ITP. “Newly diagnosed ITP” applies to the first 3 months after diagnosis, “persistent ITP” describes cases continuing from 3 to 12 months after diagnosis, and “chronic ITP” defines cases lasting longer than 12 months. Regardless of the phase of the disease, “severe ITP” refers to situations where bleeding at presentation is significant enough to mandate treatment or where new bleeding requiring escalation of therapy occurs in previously controlled cases ( ).

ITP affects all ages, races, and genders. It is the most common reason for thrombocytopenia in childhood. The typical presentation in children is an abrupt onset of petechiae and purpura in a previously completely well child who remains playful and interactive. About 60% will have had an infection (usually viral) in the preceding 3 to 4 weeks. The peak age at diagnosis is 2 to 6 years. The majority of children present with a platelet count of less than 20 × 10 ⁹ /L. However, more than 97% will have only mild bleeding, probably because their circulating platelets are fresh, large, and granular and thus very effective at hemostasis. Although severe bleeding (the most feared being intracranial hemorrhage) does occur more commonly at lower platelet counts, the correlation between platelet count (or duration of ITP) and bleeding is not uniform or predictable. Mucosal bleeding (epistaxis, oral, melena, menorrhagia, hematuria) develops more commonly in children who will subsequently suffer an intracranial hemorrhage and thus should be urgently treated. Fortunately, ITP in children resolves spontaneously within 6 months in approximately 80% of cases. The 20% of children who develop chronic ITP are usually older (older than 10 years) and/or have platelet counts greater than 20 × 10 ⁹ /L at diagnosis. Fortunately again, more than 50% of children with chronic ITP will enjoy spontaneous remission within 4 years of diagnosis ( ; ; ; ; ; ; ; ).

The diagnosis of ITP is ultimately made on clinical grounds. There are no gold-standard diagnostic laboratory tests. At initial presentation, inquiry should be made about a personal history of other medical conditions, drug usage, or recent immunizations that may be associated with secondary ITP. A history of a recent infection might prove useful, as might a family history of thrombocytopenia. Lymphadenopathy, splenomegaly, and other physical findings of malignancies or autoimmune diseases should be sought. In ITP, the complete blood count should reveal normal red blood cell (RBC) and white blood cell (WBC) counts, and the peripheral blood smear should be normal except for thrombocytopenia. Recent guidelines from the American Society of Hematology do not recommend a bone marrow examination or testing for antinuclear antibodies in newly diagnosed children with typical features of ITP, nor do they recommend routine testing for H. pylori in children with chronic ITP. Although the workup may reveal causes of secondary ITP, the diagnosis of primary ITP remains one of exclusion, with confirmation found in positive responses to appropriate management ( ; ; ; ).

The goal of treatment of ITP is not to achieve a “normal” platelet count, but rather one that is associated with adequate hemostasis. Because of the self-limited character of ITP, the low incidence of life-threatening bleeding, and the toxicities associated with treatment modalities, the American Society of Hematology recommends observation alone in children presenting with no bleeding or mild bleeding (only skin bruises and petechiae) regardless of platelet count. However, this “watch and wait” strategy relies on avoidance of risk factors (high-contact activities, use of aspirin or NSAIDs) and heightened observation for signs of significant bleeding, especially intracranial. This approach can create significant anxiety and compromise quality of life in children with ITP and their families. If bleeding, concern about the risk of bleeding, or quality-of-life issues mandate intervention in children with ITP, initial pharmacologic management may include a short course of corticosteroids, IVIG, and/or anti-Rho(D). Corticosteroids may suppress abnormal immune responses, and IVIG may impair clearance of antibody-coated platelets by the reticuloendothelial system. Anti-Rho(D) binds to Rh-positive RBCs and blocks platelet clearance by overwhelming the reticuloendothelial system with these antibody-coated RBCs. Anti-Rho(D) is thus useful only in Rh-positive patients who have not had a splenectomy. It may, however, cause an accelerated hemolysis, leading to a symptomatic anemia. Second-line therapies may be necessary if bleeding or its risk is persistent, although there is little evidence to guide their use. High-dose corticosteroids may be helpful, but their long-term use should be avoided in children because of their significant side effects. Splenectomy offers the best response of any treatment, may be curative in over 60% of cases, and may provide relief from the toxicity of other therapies. However, splenectomized patients have an increased risk of infection by encapsulated organisms with subsequent sepsis. Therefore at least 3 weeks before splenectomy, patients should be immunized against Streptococcus pneumonia, Haemophilus influenza type b, and Neisseria meningitidis. After splenectomy, they should receive oral prophylactic antibiotics and rigorous septic workups when febrile. For these reasons, splenectomy is delayed, if possible, for at least 12 months after diagnosis in hopes of spontaneous remission. Rituximab, a monoclonal antibody against the CD20 antigen on antibody-producing B lymphocytes, attenuates antiplatelet antibody production and may be useful as a second-line therapy or as an alternative to splenectomy. However, rituximab may allow reactivation of latent viruses, potentially leading to fatal hepatitis B or to progressive multifocal leukoencephalopathy (Jakob-Creutzfeldt virus). Thrombopoietin receptor agonists (romiplostim and eltrombopag) stimulate platelet production by megakaryocytes in bone marrow and may be useful when ITP relapses after splenectomy or in situations where splenectomy is not feasible. Their use may be complicated by rebound thrombocytopenia upon discontinuation, bone marrow fibrosis, thrombosis, progression of hematologic malignancies, and hepatotoxicity ( ; ; ; ; ; ; ; ; ).

Emergency treatment of ITP in cases of severe bleeding aims to increase the platelet count as quickly as possible. IVIG is especially useful in this regard and should be combined with corticosteroids, anti-Rho(D), and platelet transfusions as needed. Antifibrinolytic agents, activated recombinant factor VII, and emergency splenectomy have all been used as well. Similar therapies may be used “on demand” in preparation for surgical procedures. A platelet count of at least 20 to 30 × 10 ⁹ /L is probably adequate for low-bleeding-risk procedures, whereas counts greater than 50 × 10 ⁹ /L should be achieved for higher-risk procedures ( ; ; ).

Thrombotic thrombocytopenic purpura

Thrombotic thrombocytopenic purpura (TTP) is another condition involving platelets and belongs to a group of disorders collectively termed thrombotic microangiopathies (TMAs). These disorders are characterized by episodes of thrombocytopenia (but with normal platelets), microangiopathic hemolytic anemia, and ischemic organ damage. TTP is caused by a severe deficiency of the protease ADAMTS13 ( A D isintegrin A nd M etalloprotease with T hrombo S pondin type 1 repeats, member 13 ). ADAMTS13 is responsible for regulating vWF by cleaving ultra-large multimers of vWF as they are released into the circulation from endothelial cells. These ultra-large multimers contain an increased number of platelet binding sites and, when allowed into the circulation, induce platelet adhesion and the formation of platelet thrombi in the microvasculature. Thrombocytopenia subsequently results from consumption of platelets in these thrombi, hemolytic anemia results from mechanical fragmentation of RBCs during flow through partially occluded small vessels, and ischemic organ injury results when thrombi become occlusive. ADAMTS13 deficiency can be acquired (more common) or congenital. The acquired form is the result of circulating anti-ADAMTS13 IgG autoantibodies. The congenital form is the result of homozygous or compound heterozygous mutations of the ADAMTS13 gene on chromosome 9 resulting in a severely reduced (<10%) level of ADAMTS13. Heterozygotes have half-normal enzyme activity levels and are generally asymptomatic. It was initially thought that the acquired form was seen in adults, whereas the hereditary form occurred in children. However, each type has been reported in both adults and children (George 2010; ; ; ; ; Scully and Goodship 2014).

Up to 50% of children with hereditary ADAMTS13 deficiency (Upshaw-Schulman syndrome [USS]) will experience acute episodes of thrombocytopenia and microangiopathic hemolytic anemia during the neonatal period. Other patients with USS may remain asymptomatic until events that trigger the release of vWF are encountered, such as infections, immunizations, pregnancy, or alcohol binge drinking. Acute episodes may also include findings caused by organ ischemia, most notably involving the brain, heart, gastrointestinal tract, or kidneys. Acquired TTP is more a disease of preadolescent and adolescent children, although it can and does occur even in children less than 2 years of age. Similar triggering events precipitate acute episodes ( ; ; ).

The diagnosis of TTP should be based on history and clinical findings. Family history may be helpful in USS but will not be in acquired TTP. A “classical pentad” of thrombocytopenia, microangiopathic hemolytic anemia, fever, renal failure, and central nervous system impairment has been described, but all components are rarely seen at presentation. Thrombocytopenia (<30 × 10 ⁹ /L) and microangiopathic hemolytic anemia (with schistocytes [RBC fragments] in the peripheral blood smear) should prompt consideration of the diagnosis. USS should be considered in any neonate with thrombocytopenia and severe jaundice. The diagnosis of TTP is confirmed by finding low ADAMTS13 activity level (<10%) in plasma or anti-ADAMTS13 IgG antibodies using an enzyme-linked immunosorbent assay (ELISA) method. Gene sequencing can ultimately confirm the diagnosis of USS ( ; ; ).

Acute episodes of TTP can mimic hemolytic uremic syndrome (HUS) but must be differentiated because their treatments differ. In general, platelet counts are higher in HUS (>30 × 10 ⁹ /L), as is ADAMTS13 activity (>20%), and anti-ADAMTS13 autoantibodies are not present ( ; ; ; Scully and Goodship 2014). Acute episodes of TTP (whether acquired or hereditary) should be treated emergently with plasma exchange to replace ADAMTS13 and/or remove anti-ADAMTS13 antibodies. If plasma exchange capabilities are not immediately available, large-volume plasma infusions should be carefully undertaken. RBC transfusion may be necessary when severe anemia occurs. Platelet transfusion may be used in situations of life-threatening hemorrhage, but should not be used just to treat thrombocytopenia itself. Extreme caution is warranted if platelets are administered because of the risk of precipitation of further thrombotic events. In acquired TTP, corticosteroids may also be administered in an attempt to suppress anti-ADAMTS13 antibodies. Response to plasma exchange (with or without added corticosteroids) is judged by platelet count recovery and control of hemolysis. Further management of acquired TTP may include the use of the monoclonal antibody rituximab if thrombocytopenia persists, clinical symptoms progress, or a controlled episode of TTP relapses. Rituximab is directed against the CD20 protein on the surface of antibody-producing B lymphocytes, allowing these cells to be destroyed and thus attenuating the production of anti-ADAMTS13 autoantibody. Rituximab and/or scheduled plasma infusions may prevent relapses of TTP episodes in those with ongoing anti-ADAMTS13 antibody production or persistently low ADAMTS13 levels. Recombinant ADAMTS13 is in development, and its use in a murine model of TTP shows promise as a potential future therapeutic option. (George 2010; ; ; ; ; Scully and Goodship 2014; ).

Thrombophilias

Thrombophilia is a term describing hereditary or acquired conditions that enhance the coagulability of blood and thus predispose affected individuals to the development of venous or arterial thromboses ( ). Associated inherited conditions include deficiencies of the naturally occurring anticoagulants antithrombin (AT), protein C (PC), and protein S (PS) and “gain of function” mutations resulting in factor V Leiden (FVL) and prothrombin gene mutation G20210A. Associated acquired conditions include the antiphospholipid syndrome and a multitude of pathologic or interventional clinical situations, including sepsis, malignancies, nephrotic syndrome, trauma, surgery, indwelling vascular catheters, or use of oral contraceptive pills ( ; ).

Fortunately, the occurrence of venous and arterial thromboses is uncommon in children, although it is being recognized with increasing frequency ( ). Inherited thrombophilias that may predispose to these events are present in 5% to 10% of the general population. Heterozygosity for AT, PC, or PS deficiency; homozygosity for any of the mutations; and inheritance of multiple mutations are considered high-risk thrombophilic situations. Heterozygosity for FVL or prothrombin gene mutation G20210A is considered lower risk ( ). Most heterozygous individuals will never experience a thrombotic event, although the risk they carry is lifelong ( ; ). Acquired clinical risk factors such as the presence of indwelling vascular catheters or the use of oral contraceptive pills are often more important in provoking the development of thrombotic events in children. However, not all children with the same clinical risk factors and not all children with the same inherited thrombophilias will develop thromboses. It appears that the imposition of clinical risk factors on an individual genetic predisposition produces powerful synergy for the development of thromboses. Therefore familiarity with inherited and acquired thrombophilias is important for clinicians caring for children.

Antithrombin irreversibly inhibits primarily factor Xa and thrombin in addition to factors VIIa, IXa, XIa, and XIIa. AT circulates in blood in a quiescent form, but its inhibitory actions are phenomenally augmented by the presence of heparan sulfates from activated endothelial cells or by exogenously administered heparin. Deficiencies of AT result in reduced inhibition of factor Xa and thrombin with subsequent increased thrombin generation and activity. AT deficiencies can be quantitative (type I) or qualitative (type II) and occur as a result of numerous mutations inherited in an autosomal dominant pattern. AT deficiency is the most thrombogenic of the inherited thrombophilias, and homozygous-type mutations (quantitative) are incompatible with life. Recombinant AT concentrate is available for use in affected patients. Heparin may be ineffective in thrombosis management in some type II mutations ( ; ; ; ).

Protein C is a vitamin K–dependent plasma protein that is activated (APC) on endothelial cell surfaces by the thrombin-thrombomodulin complex generated after vascular injury. PS is a vitamin K–dependent cofactor for PC that augments the activity of APC tenfold to inactivate factors Va and VIIIa and thus regulate ongoing thrombin production. Deficiencies of PC and PS can be quantitative (type I) or qualitative (type II). A type III PS deficiency also has been described where total PS levels are normal but free PS levels (the APC cofactor) are decreased. Homozygous PC deficiency results in neonatal cerebral vein thrombosis or in neonatal purpura fulminans, a condition characterized by progressive hemorrhagic skin necrosis. Treatment includes administration of fresh frozen plasma (FFP) to replace both PC and PS or administration of PC concentrate. Heparin administration is not effective. Lifelong anticoagulation and PC supplementation are necessary in these patients. Homozygous PS deficiency is thought to be incompatible with life, and heterozygous deficiencies of PC or PS increase thrombotic risks in young adulthood. Both PC and PS deficiencies are inherited in an autosomal dominant pattern. PC deficiency is the most common inherited thrombophilia in Japan ( ; ; ; ; ).

FVL is a mutant factor V caused by the autosomal dominant inheritance of a point mutation in the factor V gene resulting in a single amino acid substitution. FVL is inactivated tenfold more slowly by APC than is normal factor V (“APC resistance”), resulting in enhanced thrombin production. Heterozygotes for FVL have a 5- to 10-fold increased risk for venous thrombosis, whereas homozygotes have a greater than 25-fold increased risk. FVL is the most common inherited thrombophilia in White populations of European descent while being rarely found in Southeast Asian, Japanese, sub-Saharan African, or indigenous Australian populations ( ; ; ).

Prothrombin gene mutation G20210A is an autosomal dominant mutation that causes a single nucleotide substitution at position 20,210 in the prothrombin gene. It results in an increased production of prothrombin. Consequently, prothrombin levels are increased by approximately 30% in heterozygotes, with thrombosis risk increased threefold to fourfold. Homozygotes have a 70% increase in prothrombin levels, with a thrombotic risk similar to that seen with homozygous FVL (25-fold increase). The population distribution of this mutation is similar to that of FVL, and it is the second most common inherited thrombophilia (behind FVL) in this group. Combined heterozygosity for FVL and prothrombin gene mutation G20210A occasionally occur with resultant synergistic hypercoagulable effects ( ; ; ; ).

Homocysteine is generated during metabolism of the amino acid methionine. The pathway of metabolism of homocysteine requires the action of an enzyme called methylenetetrahydrofolate reductase (MTHFR). An autosomal recessive mutation in the MTHFR gene causing a single amino acid substitution results in the production of MTHFR C677T, which has half of the catalytic activity of the normal enzyme. Consequently, homozygotes for MTHFR C677T have severely elevated levels of homocysteine, which can induce endothelial injury and dysfunction. Affected individuals are predisposed to the development of venous thrombotic events and strokes (ischemic arterial strokes or cerebral sinus venous thrombosis). This mutation is predominantly found in White European populations ( ; ).

Antiphospholipid syndrome (APS) is an acquired autoimmune thrombophilia caused by antibodies directed against anionic phospholipids (such as cardiolipin in mitochondrial membranes and phosphatidylserine in virtually all cell membranes) or, more importantly, against antigens that bind to phospholipids (especially beta-2-glycoprotein-I [β ₂ GPI] and prothrombin). “Lupus anticoagulant” is a term applied to anticardiolipin, anti-β ₂ GPI, and antiprothrombin antibodies. β ₂ GPI is the major antigen associated with APS, and anti-β ₂ GPI antibodies most closely associate with the clinical manifestations of APS. These clinical manifestations include arterial or venous thromboses in any organ and pregnancy complications or losses. Diagnosis of APS depends on documentation of at least one clinical manifestation and one positive laboratory test for antiphospholipid antibodies (APLAs) within 5 years of each other and persistence of positive laboratory tests for at least 12 weeks. APS is deemed secondary if associated with autoimmune disorders such as systemic lupus erythematosus or rheumatoid arthritis and primary in the absence of such association. APLAs, especially anti-β ₂ GPI antibodies, in primary APS probably develop as a result of molecular mimicry between human β ₂ GPI and similar molecules in invading bacteria. Neonatal APS can result from transplacental passage of maternal APLAs. APLAs predispose to thrombosis by inducing tissue factor expression on endothelial cells, augmenting platelet activation, impairing AT and APC control of coagulation, and inhibiting fibrinolysis. Management of initial venous thrombotic episodes usually includes a finite duration of anticoagulation. Recurrence of venous thromboses or any occurrence of arterial thrombosis calls for indefinite anticoagulation with aspirin and/or warfarin. Primary thromboprophylaxis for asymptomatic individuals with positive APLA tests is not deemed necessary ( ; ; ; ; ; ; ).

A difficult question in dealing with thrombophilias is: Who should be screened? Interpretation of thrombophilia screening tests requires expert insight. Indiscriminate use of these tests is discouraged, but the infrequency of these abnormalities makes controlled trials to determine who should be screened difficult. General guidelines based mostly on expert opinions suggest screening individuals who present with thrombotic events at an early age (<40 years) and individuals with recurrent thromboses, thrombotic events unprovoked by concurrent clinical risk factors, or strong family histories of thromboses. Long-term anticoagulation is often advised in patients with “high-risk” thrombophilias; that is, those with AT, PC, or PS deficiencies (heterozygotes or homozygotes); those homozygous for FVL; and those with multiple coexisting defects ( ; ). The anesthetic implication of thrombophilias is mainly one of awareness: awareness of their existence; of their pathophysiologic mechanisms; and of the potential for long-term anticoagulant use in order to converse and plan intelligently with patients, parents, and other physicians. The advice of an experienced hematologist is invaluable in preparing a perioperative plan for these patients.