Chapter 6 Laboratory Diagnostics in

Thrombosis and Hemostasis

Peng Liming

OVERVIEW OF HEMOSTASIS

• Primary and Secondary Hemastasis Hemostasis may be defined as the process that maintains

flowing blood in a fluid state and prevents loss of blood from sites of vascular disruption. This definition implies two major components:

The first, a potent procoagulant mechanism that is capable of forming stable hemostatic plugs at sites of vascular disruption; and second, regulatory systems that confine normal hemostatic plug formation to sites of vascular disruption.

Key structural features of normal resting platelets are depicted (Figure 6-1.)

Hemostatic function of platelets (Figure 6-2.)

Hemostatic function of blood vessel wall (Figure 6-3. )

Characters of coagulation factors(1) (TABLE 6-1. )

factor namesynthesize site

MW(104)

AAgenelength(kb)

gene location

on chromos

ome

plasma concentr

ation (mg/L)

half life(h)

function

Ⅰ fibrinogen

liver34

2964

504q31 2000-

400090

final substrate

Ⅱ prothrombin

liver 7.2

579

2111p11-

q12150~200 48-

96proprotei

nase

Ⅲtissue factor

many kindof cells

4.5

263

12.41p21-22

cofactor

Ⅴ labile factor

liver, platelet

332196

>801q23 5-10 12-

15cofactor

Ⅶ stable factor

liver5

406

12.813q34 0.5-2

6-8proprotei

nase

Mechanism of coagulation & anticoagulation (Figure 6-4.)

The formation of a hemostatic plug usually begins when the vessel wall is damaged, exposing the thrombogenic

bendothelial tissue to blood (Figure 6-5. )

Characters of coagulation factors(2) (TABLE 6-1. )

factor

namesynthesize site

MW(104)

AAgenelength(kb)

gene location

on chromos

ome

plasma concentr

ation (mg/L)

half life(h)

function

Ⅸ Christmas factor

liver 5.6

415

34Xq26.3~2

7.15 12~

24proprotei

nase

Ⅹ Stuart-Prower factor

liver5.9

448

2513q34 6-8

48-72

proproteinase

Ⅺ thromboplastin antecedent

liver16

1214

234q35 4-6

48-84

proproteinase

XII Hageman factor

liver8

596

125q33-ter 30 48-

52proprotei

nase

XIII fibrin stabilizingfactor

liver

322744

>160(a)

28(b)

6p24-25(a)1q31-

32.1(b)

2972-120

protrans-glutamina

se

Characters of coagulation factors(3) (TABLE 6-1. )

factor

namesynthe

size site

MW(104)

AA

genelengt

h(kb)

gene location

on chromos

ome

plasma concentr

ation (mg/L)

half life(h)

function

PK prekallikrein

liver 8.58.8

619

9.8

4q35 1.5-5

35proprote

inase

HMWK

high molecularweight kininogen

liver

12626

2.7

3q26-ter 7

144 cofactor

• Regulation of Hemostasis

There are multiple systems that work in a synergistic manner to regulate the extent of clot formation. They include intact endothelium, platelet (Table 6-3), fibrin clot formation (Table 6-4), and the relative important regulatory systems are platelet, fibrin clot formation which is consisted of tissue factor pathway inhibitor (TFPI), serine protease inhibitors (serpins), protein C system and the fibrinolytic system(Table 6-5, Fig 6-6).

Characters of anticoagulation factors(1) (TABLE6-2.)

name

MW(kD)

plasma concentr

ation (mg/L)

half life(h)

synthesize site

function

gene location exo

n

gene

(kb)

mRNA(kb)

PC 62 4 8~10

liver proproteinase

2q13~14

8 12 1.8

PS 69 20~25 42 liver cofactor 3 15 80 3.5

AT 58 125 61~72

liver, endothe

lium

proproteinase

1q23~25

7 13.5 1.5

TFPI 42 0.01~0.15

1~2min

liver, endothe

lium

proproteinase

2q31~31.1

9 85 1.4

Characters of anticoagulation factors(2) (TABLE6-2.)

name

MW(kD)

plasma concentr

ation (mg/L)

half life(h)

synthesize site

function

gene location exo

n

gene

(kb)

mRNA(kb)

Hc-II 65 33~90

liver, endothe

lium

proproteinase

22q11 5 16 2.3

α2-M 725 2000~3000

liver proproteinase

12p12~13

36 48 1.5

C1-INH

104 170 liver cofactor 11q11~13.1

8 17 1.2

PZ 620 1.8~3.9 60 liver cofactor 13q34 9 14 1.6

Regulation of platelet response (TABLE 6-3.)

Component Action Effect

Prostacyclin Increases cyclic AMP Inhibits aggregation

Nitric oxide Increases cyclic GMPInhibits aggregation and adhesion

ADPase Metabolizes ADP Inhibits aggregation

Endothelial glyocalyx

Electrostatic repulsion Inhibits platelet adhesion

Regulation of fibrin clot formation (TABLE6-4.)

Component Action Effect

Tissue factor pathway inhibitor

Inhibits tissue factor/VIIa Inhibits thrombin formation

Serine protease inhibitors

Neutralize thrombin, factor Xa

Inhibit thrombin formation and activity

Protein C system Degradation of factors Va and VIIIa

Inhibits thrombin formation

Fibrinolytic system Degradation of fibrin Removes excess fibrin clot

The components of fibrinolytic system (TABLE 6-5. )

factorsMW(kD)

AA plasma concentratio

n (mg/L)

half life

chromosome function

gene

(kb)

mRNA(kb)

exon

PLG 92 791 200 2.2d 6q26-27 z 52.5 2.9 19

t-PA 68 530 0.005 4.0min 8p12-11 p 32.7 2.7 14

u-PA 54 411 0.002 7.0min 10q24 z 6.4 2.4 11

PAI-1 52 379 0.01 8.0min 7q22.1 i 12.2 2.4/3.2 9

PAI-2 46/70

393 <0.005 / 18q22.1 i 16.5 1.9 8

FⅫa 80 596 30 2~3d 5q33-ter z 12 2.6 14

PK 88 619 40 / 434-35 z 22 2.4 15

HMWK 110 626 70 5d 3q27 c 27 3.2 11

α2-AP 70 452 70 3d 17p13 i / 2.2 10

TAFI 60 410 5 10min 13q14 、11

z 、 i 48 1.8 11

Fibrinolytic mechanism and degradation products (Figure 6-6. )

LABORATORY METHODS FOR EVALUATION OF PLATELET FUNCTION

A variety of laboratory techniques may be used to assess platelet function, but a limited number of techniques are necessary for most clinical situations (Table 6-6)

Laboratory techniques for evaluation of platelet function(1) (TABLEe 6-6. )

Procedure Property Evaluated Clinical Uses

Platelet count Platelet number in peripheral blood

Platelet production

Mean platelet volume

Average platelet size Assessing platelet mass, platelet production

Bleeding time Global platelet function Screen for platelet function

PFA 100 Platelet function Screen for platelet function

Platelet aggregation studies

Platelet function Delineate type of platelet dysfunction; detection of variant vWD, HIT

vWF antigen (vWF:ag)

Quantity of vWF protein in blood

Evaluation of possible vWD

vWF:Rcof Measurement of vWF functional activity

Evaluation of possible vWD

Collagen binding of vWF

Measurement of vWF interaction with collagen

Screen for variant vWD

Laboratory techniques for evaluation of platelet function(2) (TABLE 6-6. )

Procedure Property Evaluated Clinical Uses

GP Ib binding of vWF

Measurement of vWF function

Screen for vWD

Factor VIII binding to vWF

Evaluation of factor VIII binding site on vWF

Screen for Type 2N vWD

vWF multimeric analysis

Measure of vWF polymerization

Evaluation of variant vWD

Electron microscopy Platelet morphology, platelet granules

Evaluation of congenital platelet disorders

Bone marrow exam Assessment of platelet production

Evaluation of thrombocytopenia, thrombocytosis

Platelet antibody Detection of antiplatelet antibodies

Evaluation of possible ITP

Platelet survival Rate of platelet turnover Mechanism of thrombocytopenia

PF4, βTG In vivo platelet release Detection of platelet activation

Flow cytometry Expression of membrane proteins

Evaluation of platelet activation, function and diagnosis of HIT

Flow cytometry Measurement of platelet RNA

Assessment of platelet production

The Bleeding Time

The bleeding time (BT) is a commonly performed test to assess global function of primary hemostasis. A number of techniques for determining the bleeding time have been described, but the most commonly performed method is the template bleeding time(TBT) using a disposable device.

Analysis of von Willebrand Factor

Commonly used procedures to evaluate vWF include vWF antigen concentration (vWF:ag), ristocetin cofactor activity(vWF:Rcof), vWF binding to collagen or recombinant glycoprotein lb (GP Ib), the platelet aggregation response to ristocetin, and vWF multimeric analysis.

Immunoelectrophoresis was once the only practical method available for determination of vWF:ag, but enzyme-linked immunosorbant assays (ELISAs) and automated immunoassays are now available.

New classification of vWD (TABLE 6-7. )

vWDHered

ityF :cⅧ

vWF:Ag

Rcof:A

RIPAmultimers structure

Molecular basis

Type 1 dominant

↓ ↓5%~30%

↓ ↓ multimers in plasma and platelet are normal

unknown

Type 2A

dominant

↓or N ↓or N ↓↓ ↓↓ deficiency of high or medium molecular weight multimers in plasma

defects of multimers biosynthesis or increase of proteolysis sensitivity, mutation clustered within A2 domain

Type 2B

dominant

↓or N ↓or N ↓or N ↑ deficiency of high molecular weight multimers in plasma, the types of multimers in platelet are normal

high molecular weight multimers in plasma bind with platelets spontaneously, mutation clustered in A1 domain

New classification of vWD(con’t) (TABLE 6-7. )

vWDHeredity

F :Ⅷc

vWF:Ag

Rcof:A

RIPAmultimers structure

Molecular basis

Type 2M

(multimer)

dominant

multimer distribution is normal, type of satellite bands may abnormal

mutation in vWF A1 domain influence bingding to platelet GP bⅠ

Type 2N

(nomandy)

recessiv

e

medium ↓

N N N multimer in plasma and platelet may normal

missense mutation in domain binding to FⅧ

Type 3

recessiv

e

medium or obvious ↓

deficiency

or undetected

deficiency

deficiency

reduced or absent in plasma or platelet

vWF gene deletion or partly deletion, or defects in mRNA expression

Platelet Response To Agonists

Platelet aggregation studies are used to assess the response of platelets to a variety of agonists. Platelet aggregation is most commonly performed by a turbidimetric method using platelet-rich plasma but also can be performed by an impedance method that utilizes either whole blood or platelet-rich plasma (PRP).

Nomnal (C) and abnormal (Pt) aggregation responses for

ADP, collagen, epinephrine, and ristocetin (Figure 6-7.)

Flow cytometry has emerged as a powerful tool for the analysis of platelet function. Platelet activation is associated with surface expression of proteins not found on quiescent platelets. Analysis of these proteins has been used to evaluate in vivo platelet activation, which occurs in a variety of clinical settings.

Flow diagram for diagnosis of abnormalities of platelet function (Figure 6-8. ).

LABORATORY EVALUATION OF HEMOSTATIC DISORDERS

There are a number of reasons why the hemostatic system may be evaluated during the clinical management of patients. Common reasons include therapeutic drug monitoring, presurgical evaluation of hemostasis, evaluation of a possible bleeding tendency, evaluation of a possible thrombotic tendency, evaluation for the possibility of a circulating lupus anticoagulant, evaluation for the possibility of disseminated intravascular coagulation, and evaluation for other specific disorders.

Routine presurgical evaluation of hemostasis remains a controversial topic. In general, routine preoperative bleeding times, prothrombin times (PTs) and activated partial thromboplastin times (APTTs) do not accurately predict the risk of bleeding during surgery.

Laboratory evaluation of hemostasis is indicated whenever the clinical and/or medical history suggests the possibility of altered hemostasis. As hemostasis is a balanced system with potent procoagulant and regulatory mechanisms, bleeding or thrombotic disorders can arise whenever this balance is disturbed.

Evaluation of a Potential Bleeding Disorder

• The clinical history of a patient with a potential bleeding disorder is used to determine whether the patient truly does have a bleeding problem, whether the problem is likely to be congenital or acquired, and whether the defect is related to altered primary hemostasis, secondary hemostasis, or regulation of hemostasis.

• Following the clinical history, routine screening tests are commonly performed. These include the platelet count and bleeding time to assess platelet function and the PT and APTT to assess fibrin clot formation. Note that these screening tests do not assess fibrin stabilization or fibrinolysis.

The PT evaluates the extrinsic system of coagulation, beginning with activation of coagulation by tissue factor/VIIa, and is sensitive to defects in fibrinogen, prothrombin, factor V, factor X,and factor VII.

APTT evaluates the intrinsic system of coagulation and is sensitive to defects in fibrinogen, prothrombin, factor V, factor X, factor VIII, factor IX, factor XI, factor XII, prekallikrein, and HMW kininogen.

Common patterns associated with bleeding disorders

Based on the results of the clinical history and screening laboratory tests, a limited number of patterns emerge (Table 6-8).

Platelet DisordersCoagulationDisorders

Fibrinolytic Disorders

Clinicalhistory

Mucocutaneous bleeding

Soft-tissuebleeding

Delayed bleeding

Screening

laboratory tests

Long bleeding time and/or low platelet count

Long PTand/or

APTTNormal

Laboratory Analysis of Coagulation

The PT and APTT provide an arbitrary dissection of coagulation into the intrinsic (APTT) system and the extrinsic (PT) system. Although in reality this division is artificial, it is very useful from the standpoint of patient evaluation.

Diagnosis of inherited coagulation factor deficiencies (Figure 6-9.)

The thrombin time (TT) is a very useful test and it is often overlooked. TT may be abnormal in a variety of clinical situations. TT is extremely useful in ruling out the presence of heparin in a patient sample. The finding of a prolonged TT that corrects with the addition of protamine is virtually diagnostic of heparin.

Evaluation of A Potential Thrombotic Tendency

As with the evaluation of bleeding disorders, the evaluation of a potential thrombotic tendency begins with a thorough medical history. One of the primary goals of the history is to determine the likelihood of a congenital defect.

There are no screening assays that assess the overall function of the regulatory system of coagulation or the degree of activation of the procoagulant systems. Therefore, the laboratory approach to potential thrombotic disorders involves measuring selected individual components of the regulatory systems.

Evaluation of D-dimer in the diagnosis of suspected DVT & PE (Figure 6-10.)

COAGULATION ABMORIVALITIES

• Hereditary Disorders Of Coagulation Proteins

Hemophilia A (Factor VIII Deficiency)

Hemophilia A is an X-linked inherited disorder of factor VIII (Antihemophiliac Globulin). The incidence is approximately 1:5,000 males. As previously discussed, factor VIII is a critical cofactor in the intrinsic coagulation pathway at the level of factor X activation.

The laboratory diagnosis of hemophilia A is relatively straightforward. In the majority of cases, APTT will be prolonged while PT and BT will be within normal limits. Factor VIIIC assays are necessary to establish the diagnosis. Patients with severe hemophilia A will have factor VIIIC levels, which are less than 1% (0.01 U/ml).

• Hemophilia B (Factor IX Deficiency) Hemophilia B is also a sex-linked disorder.

Christinas disease and factor IX deficiency are frequently used synonyms. The incidence of hemophilia B is approximately 1:40,000 to 1:50,000 population. However, in certain groups such as the Amish and East Indians, hemophilia B occurs as often as hemophilia A. Varying degrees of hemophilia B have been recognized with severe <l% (0.01U/ml), moderate <5% (0.01 to 0.05 U/ml) and mild >5% (>0.05 U/ml) phenotypes analogous to factor VIII deficiency.

• Hemophilia C (Factor XI Deficiency) Factor XI deficiency is an incompletely

autosomal-recessive hereditary disorder. The majority of factor XI deficiency occurs in individuals of Jewish descent. In an extensive study by Seligsohn of Ashkenazi Jews, the frequency of homozygotes for factor XI deficiency was 0.1% to 0.3% whereas the heterozygotes were found in 5.5% to 11.0%.

• Afibrinogenemia And Hypofibrinogenemia

Afibrinogenemia is a rare disorder inherited in an autosomal-recessive pattern. There often is a variable history of clinical bleeding.

Laboratory abnormalities of all of the standard screening tests are present. No end point is detected with the PT, APTT, or thrombin time. The bleeding time may be slightly prolonged and platelet aggregation studies typically show a lack of response to the usual agonists (ADP, epinephrine, and collagen).

Hypofibrinogenemic patients are characterized by a fibrinogen level, which is less than 100 mg/dl. Often these patients have a mild bleeding tendency with an autosomal dominant or, in some cases, an autosomal-recessive pattern of inheritance. In many cases, hypofibrinogenemic patients will have normal PT and APTT results. However, typically the thrombin time will be abnormal. Clottable and immunologic fibrinogen assay results are decreased thus establishing the diagnosis.

• Dysfibrinogenemia Hereditary dysfibrinogenemia is inherited in an autosornal-dominant pattern.

These abnormal fibrinogen molecules are caused by mutations, which result in single arnino-acid alteration in one of the three fibrinogen chains (a-alpha, b-beta, or gamma chains).

Laboratory findings include normal or only minimally prolonged values for PT and APTT. Typically, the thrombin time is prolonged as is the reptilase time. The abnormal thrombin time usually cannot be corrected by the addition of proramine or calcium. Laboratory diagnosis relies on the demonstration of a discrepancy between the level of clottable fibrinogen and antigenic fibrinogen.

• Acquired Disorders Of Coagulation Proteins

Oral Anticoagulant Therapy

Oral anticoagulant therapy is based on administration of coumarin or its derivatives. This class of drugs blocks the reductase enzyme in the vitamin K pathway resulting in increased levels of nonfunctional vitamin K epoxide.

The monitoring of oral anticoagulant therapy has relied upon the PT and variants of the PT(INR). For many years there has been an international controversy regarding the optimal test system tomonitor oral anticoagulants.

In order to report an INR value, it is necessary to know the International Sensitivity Index (ISI) for the thromboplastin being used as well as the geometric mean of the PT range.

The introduction of the INR has emphasized the need for lower doses of oral anticoagulants. In the past when PT results were reported as ratios, the therapeutic range was typically quoted as PT ratios of 1.5 to 2.5. With the use of the INR, it is now evident the majority of patients are satisfactorily anticoagulated with INRs of 2.0 to 3.0. As a consequence, it is anticipated the incidence of bleeding in patients receiving oral anticoagulant therapy will significantly decrease.

Disseminated Intravascular Coagulation (DIC)

DIC may be encountered in a variety of different clinical situations.

Clinical examples would include amnionic fluid embolus, head injuries, neurosurgery, and certain malignancies. Some tumor cells appear to have a specific enzyme (cysteine protease), which will directly activate factor X. Alternatively with endothelial injury, there may be activation of the extrinsic pathway leading to DIC.

In acute DIC, the constellation of laboratory findings reflect both the consumption of coagulation proteins as well as the presence of plasmin. Typically, the APTT, PT, and TT will be prolonged. In addition, there is consumption of platelets as well as the regulatory proteins AT and protein C. Fibrinogen/fibrin degradation products will be increased and the level of fibrinogen will be decreased.

In chronic DIC, the platelet count will typically be slightly decreased whereas the coagulation assays (APTT, PT) may be within normal limits. Fibrin/fibrinogen degradation products and D-dimer will be elevated and there may also be consumption of AT.

Liver Disease

Hemostatic problems encountered in patients with liver disease usually are complex. Because the liver plays a key role in the synthesis of coagulation proteins, regulatory proteins and factors involved in the fibrinolytic system, the balance between these various components may be altered to varying degrees in any patient with significant compromise of liver function.

Levels of the vitamin K dependent proteins are also decreased in liver disease associated with obstructive jaundice or malabsorption of vitamin K. Since factor VII has the shortest half-life of the vitamin K-dependent proteins, the first laboratory abnormality encountered may be an isolated prolongation of the PT.

Patients with liver disease often show a hypofibrinogenemia together with elevation of fibrin/fibrinogen degradation products and thrormbocytopenia. This constellation of laboratory findings may be difficult to differentiate from DIC.

Often in advanced liver disease there is low-grade intravascular coagulation making the differential diagnosis of DIC and liver disease an exercise in futility.