Hemostasis & White Blood Cell Disorders

OBJECTIVES

By the end of this session the reader should be able to:

  • To describe the elements required to form a platelet plug
  • To describe the coagulation cascade
  • To understand the differences between prothrombin time, partial thromboplastin time and international normalized ratios
  • To understand how to interpret white blood cell count in inflammation and leukemia

KEY TERMS

Aplastic anemia - diverse group of bone marrow disorders characterized by pancytopenia due to reduction of hematopoetic marrow stem cells

B cell lymphocyte - responsible for humoral immunity, makes plasma cells that produce antibodies

Basophil - readily stained with basic dyes, leukocyte with pale staining nucleus usually consisting of two lobes and prominent blue granules containing vasoactive amines such as histamine and serotonin

Coagulation - enzyme cascade to form a fibrin clot

Eosinophil - a leukocyte with prominent red granules and a nucleus with two lobes, readily stained with eosin

Granular leukocytes - leukocytes with abundant granules in cytoplasm (neutrophils, eosinophils and basophils)

Hemostasis - the process of stopping bleeding

Hypoplastic anemia - general term indicating a form of anemia due to varying degrees of erythrocytic hypoplasia

Leukemia - progressive malignant disease of the blood - forming organs, characterized by distorted proliferation of leukocytes and their precursors

Leukocyte - white blood cell

Leukopenia - reduction in number of leukocytes in blood (< 5000/mm3)

Lymphocyte - immunologically competent cells, divided into T and B cell lines

Lymphoma - malignant disease of lymphocytes, producing enlarged lymph nodes and spleen

Lymphopenia - reduction in number of lymphocytes

Monocyte - a mononuclear phagocytic leukocyte

Neutropenia - a decrease in the number of neutrophilic leukocytes in blood

Neutrophil (polymorphonuclear leukocyte, segmented leukocyte) - a granular leukocyte having a nucleus with 3 to 5 lobes, having properties of chemotaxis, adhering to immune complexes, and phagocytosis; these cells are the most important in defense against bacterial infection

Nongranular leukocytes - leukocytes without granules in cytoplasm (lymphocytes and monocytes)

Pancytopenia - deficiency of all cellular components of blood

Reticulocyte - a young red blood cell showing basophilic reticulum (rough endoplasmic reticulum on which hemoglobin is synthesized)

T cell lymphocyte - responsible for cellular immunity, T helper, cytotoxic and suppressor cells

Thromobocyte - a blood platelet

Thrombocytopenia - reduction in number of platelets

von Willebrand’s disease - autosomal dominant hemorrhagic disease caused by lack von Willebrand factor (vWF) and low coagulation factor VIII which is carried on vWF

BACKGROUND SIGNIFICANCE

Hemostasis is essential for life and is tightly controlled through complex processes including formation of a platelet plug, activation of the coagulation cascade and through vasoconstriction. Many diseases and drugs affect hemostasis and the hematology laboratory provides key information to diagnose, treat and monitor coagulopathies. In addition to hemostasis, the hematology laboratory also provides quantitation of a variety of white cells and precursors essential for detecting disease and/or drug-induced changes to leukocytes.

HEMOSTASIS

Platelet Plug (See Figure 1)

A) With tissue injury, platelets adhere to vWF through GPIb-IX-V, vWF adheres to subendothelial matrix.

B) Platelets adhere to collagen through GPIa-IIa.

C) Platelets aggregate when GPIIb-IIIa binds to fibrinogen after platelets are activated (thrombin, ADP, collagen are activators).

D) Activated platelets secrete ADP, and thromboxane (which causes vasoconstriction).
Activated platelets synthesize and display factor Va on their surfaces.

formation_plateletplug_1.jpg

Figure 1: Formation of a Platelet Plug

Coagulation Cascade

A sequence of proteolytic cleavages of preformed enzymes that eventually leads to thrombin cleaving peptides from fibrinogen, forming fibrin which polymerizes.

Platelet plug initiates and is required for coagulation cascade

Form a fibrin clot

  1. Injured endothelium makes tissue factor (TF)
  2. TF clips and activates circulating factor VII
  3. Factor VIIa activates circulating X and IX
  4. Xa binds to Va on platelets, localizing enzyme complex that converts II (prothrombin) to IIa (thrombin)
  5. IIa converts fibrinogen to fibrin which polymerizes to form a clot
  6. XIII crosslinks fibrin to form stable clot

Backup and amplifying systems

  1. IXa complexes with VIII carried on vWF to form IXaVIIIa
  2. IIa activates more VIIIa and Va, XIa
  3. Collagen activates circulating XII into XIIa which activates XI into XIa which upregulates IX into IXa
  4. IIa also activates XI into XIa

Moderating factors that localize clot

  1. Thrombomodulin on endothelial cells, changes the activity of factor II causing it to activate protein C. Protein C combined with S inactivate V and VIII.
  2. Antithrombin III, a circulating molecule is activated by cell surface heparan (same molecule as circulating heparin). When heparan is activated it inhibits factors II and X.
  3. Plasmin, which is activated by the factor XII complex, digests fibrin clots eventually releasing d-dimer.

Prothrombin Time (PT)

  • Measured in seconds (normal approximately 10 seconds)
  • Time for conversion of prothrombin to thrombin
  • Evaluates the factor VII dependent pathways of the coagulation cascade
  • Performed by adding tissue factor (TF) to plasma
  • TF (+ platelet phospholipids, Va) → VIIa → Xa Va → IIa → I → clot
  • Often converted into International Normalized Ratio (INR) which is (patient PT/Control PT)ISI where the ISI is the international sensitivity index (a measure of the reagents sensitivity to VIIa); normal INR is 0.9-1.5
  • PT’s are sometimes difficult to compare between laboratories, INR can be directly compared
  • Best for monitoring coumadin therapy (which primarily inhibits synthesis of VII as well as II, IX and X)
  • Patients on anticoagulant drugs should have an INR of 2.0 to 3.0 for basic “blood-thinning” needs
  • For some patients who have a high risk of clot formation, the INR needs to be higher (2.5 to 3.5)

Partial Thromboblastin Time (PTT, also APTT)

  • Measured in seconds (normal is 23 to 33.5)
  • Time for clot to form
  • Evaluates the factor XII dependent pathways
  • Performed by exposing blood to negatively charged surface
  • Negative surface → XIIa → XIa → IXa, VIIIa → Xa Va → IIa → I → clot
  • Best for monitoring heparin which mainly inhibits II; it also inhibits X, XI, and XII because IIa amplifies these factors
  • PTT is set screen for hemophillia A and B

Other Lab Tests

  • Don’t analyze for all cofactors
  • Mix patient sample with commercially available plasma that is devoid of specific cofactors and see if clotting occurs; if coagulation does not occur then can isolate missing cofactor
coagcascade_2.jpg

Figure 2: Coagulation Cascade

Anticoagulant Drugs

Heparin

  • Requires antithrombin III (ATIII) for anticoagulant effects
  • With ATIII inhibits thrombin (Factor II), IXa and Xa
  • Uses include treatment for: venous thrombosis, pulmonary embolism, initial management of unstable angina and acute myocardial infarction, coronary angioplasty and cardiac bypass surgery

Warfarin

  • Antagonist of Vit K
  • Inhibits carboxylation of factors II, VII, IX and X, thus preventing these coagulation factors from becoming active
  • No effect on circulating, active forms of the above coagulation factors, therefore delayed onset of action
  • Also inhibits synthesis of factors C and S so may paradoxically enhance clotting in some patients

Thrombolytic Drugs

Streptokinase

  • Forms non-covalent attachment with plasminogen activating it to form free plasmin
  • Free plasmin digests fibrin clots

Tissue Plasminogen Activator (t-PA)

  • Activates plasminogen bound to fibrin
  • Reteplase is a deletion mutant of t-PA

Antiplatelet Drugs

Aspirin

  • Acetylates serine residues on cyclooxygenase
  • Prevents formation of thromboxane A2
  • Thromboxane A2 causes vasoconstriction and platelet aggregation

Clopidogrel (Plavix)

  • Inhibits platelet aggregation by blocking purine receptor
  • May cause thrombotic thrombocytopenic purpura (TTP)

Abciximab (Reopro)

  • Fab fragment of humanized monoclonal antibody directed against IIb/IIIa receptor

Platelets

  • ↑ in inflammation, disseminated cancer
  • ↓ idiopathic thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), bone marrow failure
  • ↓ in disseminated intravascular coagulation (DIC)

WHITE CELLS

wbc_3.jpg

Figure 3: White Blood Cells

White Blood Cells

  • ↑ in infection and inflammation
  • ↑, −, or ↓ in leukemia
  • Key difference is that leukemia has immature cells
  • ↓ in aplastic anemia (often drug-induced), often do not recover
  • ↓ in hypoplastic anemia, often recover after drug stopped

Neutrophils

  • ↑ with steroid administration
  • ↑ with inflammation (bacterial infection)
  • Variable effect with leukemia, sometimes greatly ↑, particularly immature cells

Lymphocytes

  • ↑ with viral infections
  • ↓ with steroid administration, immune deficiency

Neutropenia, Lymphopenia, & Thrombocytopenia are often drug-induced

  • Not always cytotoxic chemotherapy
  • Immune mediated or idiosyncratic
  • Stop drug
  • One cell line decreased usually indicates drug-induced vs. all cells decreased usually indicates a stem cell problem (disease or drug-induced)

CASE STUDIES

Case 1

(Ann. Pharmacother. 37(2):212-215, 2003)

A 71-year-old woman with numerous medical conditions, including type 2 diabetes mellitus, atrial fibrillation, hypothyroidism, congestive heart failure, and gastroesophageal reflux disease, was enrolled in an anticoagulation clinic where telephone follow-up of prothrombin time/international normalized ratio (INR) is performed. She was anticoagulated with warfarin (24 mg/wk) for atrial fibrillation and had a stable INR of 2.1. Several weeks after starting fenofibrate (200 mg/d) her INR was noted to be 6.7 during routine monitoring. Upon telephone interview, her caretaker denied that the patient had recent alcohol intake and any changes in medicines, health status, or diet.

  1. How do you interpret the change in this patient’s INR?
  2. What other laboratory tests might be helpful in the management of this patient?
  3. What dose recommendations would you suggest?
  4. Propose a mechanism for this interaction.
  5. Are interactions common with these kinds of drugs?

Case 2

(Therapeutics & Toxins News 18(1):1-3, 8-10, February 2003)

A 19-year-old 70 kg healthy male presented to ED with concerns he had been poisoned at a restaurant with pellets that resembled d-Con Mouse Prufe-IIO. A computer search indicates that the active ingredient is brodifacoum. Lab results include a PT of 11.3 seconds and an INR of 1.2. The patient is discharged without treatment. Two days post exposure the patient again presents to a different ED complaining of stomach pain and blood in his stool. Lab results include a PT of 12.5 seconds and an INR of 1.2. Again the patient is discharged without treatment. Eight days post exposure the patient presented to a clinic complaining of abdominal discomfort, dizziness, bloody stool and nosebleeds. Lab results include a PT 94.7 seconds, INR 9.2, and Hct 42.2%.

  1. How do you explain these symptoms and laboratory findings?
  2. Why did it take days before the PT and INR became elevated?
  3. Why is the Hct not low?
  4. What is the mechanism of action of this rodent poison?
  5. What is the appropriate therapy for this patient?
  6. How long will this patient need to be treated?
  7. Is this a common type of poisoning?

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