CLINICAL (7.

2P) Platelets, Blood Coagulation and Fibrinolysis Disorders; Leukocytic Disorders

PATHOLOGY
Dr. Myrna Espiritu| Feb 16
Prelim Quiz 2

LAB TESTS TO DIFFERENTIATE RELATIVE POLYCYTHEMIA FROM PV IV. ESSENTIAL THROMBOCYTOSIS

Proliferation of megakaryocytic elements
Increased Platelet count
Diagnosis of Exclusion( not common: R/O CML first, CML also has increased
Blood volume decreased total Increased RBC mass platelet count at initial stage)
measurements (to blood
know whether the volume(plasma V. LEUKEMOID REACTION
Polycythemia you are volume) & Abnormally marked granulocytic response to some BM stimulus, commonly
seeing in your patient is normal RBC infection
due to an increase in RBC
mass Best differential
mass or a decrease in Non-neoplastic
plasma volume) Peripheral Blood Smear
-WBC count: 50,000-100. 000/cumm( marked leukocytosis)
arterial blood decreased Normal because it is not a secondary
response -Shift to the left- extend to myelocytes
saturation studies
o No basophilia( good differentiating factor)
(oxygen)
HYPOXIA (most common Bone marrow
cause of secondary Myeloid hyperplasia with immaturity
polycythemia) Not as great as CML
Leukocyte alkaline Elevated in 90% of patients (regardless DIFFERENTIAL DIAGNOSIS OF CML, AMM, LEUKEMOID REACTION
phosphatase (LAP) of the WBC count) PBS:
- Very High WBC count: more likely CML
BMA biopsy not always diagnostic -Basophilia: CML>PM, not seen in LR
(Bone marrow hyperplasia with elevated counts of all -Tear drop cell: PM>CML, not in LR
elements , there is always hyperplasia so it should be BM:
correlated with other tests) -CML: hypercellular marrow with moderate immaturity
Erythropoietin Normal: Relative erythropoietin levels decreased -LR: some but lesser degree
hormone assay PV (not dependent on EPO, remember that we -PM: fibrosis
some causes of are talking about neoplasia, and neoplasia LAP: Leukocyte alkaline phosphatase
Increased:
secondary/relative is an autonomic growth, it may decrease -LAP granules are found in mature and moderately immature neutrophil
Secondary PV
polycythemia vera would EPO as a negative feedback) >LR: score is higher than reference range
be related to increase >PM: variable
levels of erythropoietin >CML: score is below reference range
(EPO)-hormone and is a -Score: 0- no granules
trigger factor for 1+- occasional diffuse granules
development or 2+- moderate number of diffuse granules
elevation of RBC mass
3+- many strongly positive granules
serum uric acid level Elevated
4+- confluent shows positive granules
(Due to Increase red cell turn over or
destruction of blood elements)
 LYMPHOID NEOPLASM Fatigue due to anemia
Fever (absence of mature leukocytes)
Classification of ALL: Bleeding (secondary to thrombocytopenia)
L1: Small homogenous blasts; mostly in children o Bone pain and tenderness
L2: Large heterogenous blasts; mostly in adults
o LAD, splenomegaly, hepatomegaly
L3: Burkitt large basophilic B cell blasts with vacuoles
o CNS manifestations (Headache, vomiting, nerve palsies)
WHO classification of Myeloproliferative syndrome (MPS) Factors associated with a worse prognosis:
Age less than 2

(7.2P) CLINICAL PATHOLOGY: Platelets, Blood Coagulation and Fibrinolysis Disorders; Leukocytic Disorders
*Precursor B lymphoblastic leukemia/lymphomaALL in children (80-85% of childhood ALL);
lymphoma in young adults and rare; FAB L1 and L2 blast morphology Presentation in adolescence or adulthood
*Precursor T ALL-15% of childhood ALL and 25% adult ALL PBS blast count greater than 100,000
*Burkitt Leukemia/Lyphoma (FAB L3) Unfavorable genetic alterations: t(9; 22)
Philadelphia chromosome is not just diagnostic but also a guide for
Acute Lymphoblastic Leukemia/Lymphoma (ALL) treatment

Immature pre-B or pre-T lymphocytes (Lymphoblasts) MATURE CELL NEOPLASM

Precursor B cell tumors A .CHRONIC LYMPHOCYTIC LEUKEMIA
o Usually present as childhood acute leukemia exclusively in elderly
Precursor T cell tumors Lymphocytosis unrelated to viral infection
o Lymphoma in adolescent males hypermature lymphocytes with highly condensed nuclei
30% of all leukemia, uncommon among Asians
Certain overlaps that make them morphologically indistinguishable malignancy of B lymphocytes
Immunophenotyping (differentiates ALL from AML) LAB FINDINGS
o Tdt (Terminal deoxytransferase): marker of immaturity total WBC count: elevated
CD8: T cell Mild-moderate N/N anemia, usually w/o reticulocytosis
CD20: B cell Platelets: decreased in 40% of cases
Cytogenetic and molecular markers: BM: 20% lymphocytes at least
o Hyperploidy:
PBS: lymphocytosis
o Polyploidy
CLINICAL FINDINGS
o Translocations t(12;21) t(9;22)
Splenomegaly
9;22:
LAD
Also seen in CML (Philadeplphia chromosome)
Hepatomegaly
Indicates Poor prognosis/ unfavorable
considerable tendency to chronic inflammation( and infection) often
12;21:
becomes the cause of death
Indicated Good prognosis/ favourable
tendency to develop autoimmune haemolytic anemia
o hypogammaglobulinemia( function of B cells, which is production of
Clinical Features
gammaglobulins, is compromised)
Common in both ALL and AML:
MAJORITY:
o Anemia
o Benign and slowly progressive over a period of several years
o Neutropenia
o 20%: more aggressive
o Thrombocytopenia
o Increased chance of developing a second malignancy
More common in ALL: o Richters syndrome: 5% of CLL evolve into NHL
o Abrupt stormy onset
o Symptoms related to depression of BM function
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 B. HAIRY CELL LEUKEMIA Factors in coagulation
Clinical variation of CLL 1. Endothelial damage (most important)
B lymphocyte disorder Exposure of collagen highly thrombogenic
Splenomegaly is a common finding 2. Von Willebrands Factor
Laboratory Picture: Formation of primary plug
Cytopenia of one or more cell lines
Binds platelets together
Hairy cells : similar to a lymphocyte in appearance but the cytoplasm
Platelets: release granule contents
appears formed, or has irregular hair-like projections

(7.2P) CLINICAL PATHOLOGY: Platelets, Blood Coagulation and Fibrinolysis Disorders; Leukocytic Disorders
o Peripheral blood o Activation of multiple coagulation factors
o Spleen o Fibrin: end product
o Bone marrow diagnostics Main component
Contain an acid phosphate which is resistant to inhibition by L-tartrate( Primary hemostasis: platelets (platelet plug)
TRAP- tartrate-resistant Acid phosphatase) Secondary hemostasis: coagulation factors (fibrin)

Factor XII: capable of autoactivation if exposed to collagen/ platelet plug

Factors that inhibit thrombosis:
Anti-thrombin
Protein C and S
TFPI (Tissue Factor Pathway Inhibitor)
NO
Components: Fibrinolytic cascade
Blood vessels
Platelets COAGULATION CASCADE
Coagulation factors Activation of a complex series of reactions involving development of
Requires the participation of cellular components proteins (platelets endothelial cells, enzymes from their precursors
neutrophils and monocytes) and plasma plasma protein (coagulation factors and PATHWAYS
regulator of coagulation e.g naturally-occurring anticoagulant and fibrinolytic system) INTRINSIC PATHWAY
Endothelial cells Stimulus: activation of Factor XII activates (Factor XI to Factor Xia) (factor
- Has procoagulant and anticoagulant properties IX to Factor IXa) (factor X to Factor Xa) prothrombin thrombin
(COMMON PATHWAY)
EXTRINSIC PATHWAY
Stimulus: Tissue damage release tissue factors activation of Factor VII
Factor VIIa activation of Factor IX Factor IXa activate Factor X
prothrombin thrombin (COMMON PATHWAY)
Thrombin
Product of both intrinsic and extrinsic pathway
Major physiologic activator of platelets together with other agonists of
coagulation
inactivated platelets : circulate without promoting coagulation
activated platelets : membranes; site for additional thrombin formation
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GOAL: Formation of thrombin PROTEINS OF THE PLASMA COAGULATION SYSTEM

1. Surface- bound zymogens

2. Vitamin K- dependent zymogens
Formation of zymogens
Complication of vitamin K deficiency: bleeding
3. Cofactors and substrate

(7.2P) CLINICAL PATHOLOGY: Platelets, Blood Coagulation and Fibrinolysis Disorders; Leukocytic Disorders
accelerate reaction in which they participate
Factor VIII (Antihemophylic Factor)
Factor IX to activate Factor X
Absence of factorVIII: Hemophilia
Factor V
Prothrombin - thrombin
Factor X activation
Fibrinogen:
main substrate of thrombin
Principal adhesive for platelet adhesion

REGULATION OF COAGULATION
FIBRINOLYTIC SYSTEM
Limits the size of fibrin clot
Accomplished by the generation of plasmin (most important)
Plasminogen plasmin
Plasminogen activation
Plasmin: dissolves fibrinogen into smaller products

REGULATORY PROTEINS
Antithrombin
Protein C & S
Tissue factor pathway inhibitor
1. Antithrombin
Inhibits the following hemostatic enzymes (IIa, Xa, VIIa, Xa, Xia,
kallejrein, XIIa) (mainly Factor IIa and Xa)
The ability of antithrombin to exerts its anticoagulation effect is
potentiated by heparin( 1000x fold more effective as an inhibitor of
coagulation)

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 2.Protein C Clot Retraction time
Activated by thrombin How long does it takes for the primary clot to be formed
Binds to thrombomodulin Tourniquet test
Inactivates factor V and VIIIa Applying pressure on forearm
Assisted by Protein S Assessment for:
3.Protein S - Defect in capillary walls
Receptor or co-factor - Thrombocytopenia

(7.2P) CLINICAL PATHOLOGY: Platelets, Blood Coagulation and Fibrinolysis Disorders; Leukocytic Disorders
Allows Protein C to bind to surfaces of cells to orient itself and inactivate Bleeding Time
Factor V and VIIIa Platelet counts <100,000/cumm
Needed by protein C o Rough correlates with degree of thrombocytopenia
Tissue Factor Pathway Inhibitor Crude test
Most potent inhibitor of the Factor VIIa Tissue Factor Complex Standardized incision: 1mm long
Valuable test for disorders involving primary hemostasis
Measure the time required for the blood to stop
More of a platelet test than a coagulation test
Normal in hemophilia A & B
***Prolonged bleeding time but normal platelet countlook for qualitative
defect in platelet
***Prolonged BT75,000/uL platelet count
Disadvantages: highly operator dependent
Use: Bleeding Time and Surgery
1.5x the upper limit: increase possibility of hemorrhage
2x the upper limit: definite Risk for haemorrhage
Determine if the patient will bleed intraoperatively

TESTS FOR PLATELET AGGREGATION

Turbidimetric Test
Sample: Patient-rich plasma
TESTS FOR PLATELET NUMBER AND FUNCTION inverse correlation
platelets aggregate: solution becomes clearer/ less turbid
Platelet count (quantitative / Smear)
Accurate: quantitative (use of machines-low specificity) Ristocetin Test
Smear: identify abnormal forms (E.G giant platelets in ITP; presence of platelet induce platelet aggregation by binding to Von Willebrand Factor to the
clumping) platelet glycoprotein complex when ristocetin is added
Platelet satellitism : platelets adherent to neutrophils (in some cases where Important test in the diagnosis of Von Willebrand disease
platelet number is normal but patient is bleeding)

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 TESTS FOR COAGULATION FACTORS 3. Liver function test- II, VII, IX, X produced by the liver
2 major conditions affecting the extrinsic System
- Liver parenchymal disease
- Vitamin K deficiency
Activated Partial Thromboplastin Time
Routine screening of coagulation disorders of the intrinsic system
Detects presence of circulating anticoagulants- antiphospholipid antibody

(7.2P) CLINICAL PATHOLOGY: Platelets, Blood Coagulation and Fibrinolysis Disorders; Leukocytic Disorders
syndrome
Monitor heparin therapy

Thrombin time
measures the availability of functional fibrinogen

Assays for Specific factors

Factor VIII assay- Hemophilia A

Clotting Time
The length of time required for a measured amount of blood to clot under
certain specified conditions
Screening test to measure all stages in the intrinsic and common
coagulation system
Monitor heparin therapy (Heparin inhibits intrinsic pathway)
Disadvantages:
1. Poor reproducibility: Operator-dependent
2. Test is sensitive only to extreme factor deficiency (false negative results)
3. Insensitive to high doses of heparin
Prothrombin Time
Screening test for the extrinsic coagulation mechanism including common
pathway ( Factors II, VII, IX, X)
Used to:
1. Monitor anticoagulation therapy (warfarin/Coumadin-given to patients with
thrombotic events; its action is to interfere with extrinsic Pathway)
antidote for warfarin/ Coumadin toxicity: Vitamin K
a. repeat prothrombin time: giving the drug in excess will cause
hemorrage,if not enough treatment will not be effective
ANTICOAGULANT SYSTEMS
1) Warfarin / Coumarin anticoagulant
Inhibits vitamin K utilization by the liver
In order of decreasing sensitivity:
o VII (most sensitive), IX, X, II (least sensitive)
For prophylaxis and/or treatment of venous thrombosis and its extension,
and pulmonary embolism
For prophylaxis and/or treatment of the thromboembolic complications
associated with atrial fibrillation and/or cardiac valve replacement
To reduce the risk of death, recurrent myocardial infarction, and
thromboembolic events such as stroke or systemic embolization after
myocardial infarction
Monitoring:
o Prothrombin time
o Previously: 2-2.5x the normal control value
o At present: use of INR (International Normalized Ratio)
2) INR
Corrects for any inconsistencies in the control samples
It is a more accurate expression of the action of warfarin
And it is currently recommended that the INR be used as the number
to adjust the dose of warfarin
Computation
INR = (Patient Protime in Seconds__________)
ISI

2. Screening for coagulation disorders-factor is deficient (II, VII, IX, X) (Mean of the Normal Protime in Seconds)
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 o ISI: International Sensitivity Index; close to 1.0
Most indications:
o INR = 2.0-3.0 (standard)
o High risk patients with mechanical heart valves: 2.5 to 3.52
o Ex. INR of 7 (stop warfarin, give Vit K)
Clinical manifestations:
Hemorrhagic disorder- patient bleeds for a long time after an injury
Hemarthrosis- bleeding within joints, knees, ankles, brain
May have nervous involvement

3) HEPARIN-ANTITHROMBIN III Treatment:

Antithrombin III (anticoagulant) is a serine protease which requires heparin as Give the deficient factor (Factor VIII)- main treatment

activator
heparin potentiates activity of AT III a thousand fold
Decrease AT III: predispose to thrombosis (due to lack of naturally occurring
anticoagulant like ATIII and protein C, which is also a reason for thrombotic
diseases)
heparin treatment: for acute MI, pulmonary embolism, DVT, DIC
monitoring of heparin Treatment:
-because AT III affects the intrinsic pathway we measure aPTT
o aPTT: 1.5-2.5 x the upper reference limit
o Get baseline of aPTT before starting treatment to know if there is a
contraindication for heparin treatment because a prolonged aPTT may indicate
a coagulation disorder.
(7.2P) CLINICAL PATHOLOGY: Platelets, Blood Coagulation and Fibrinolysis Disorders; Leukocytic Disorders
Give supportive treatment in the form of pRBC due to prolonged bleeding
**must avoid instances that could cause bleeding
**patients usually go in and out of the hospital for Blood Transfusion,
because factor VIII supplementation is eventually consumed and is not
readily available in all hospitals. Thus, they are also at risk for transfusion
related disorders.
Screening:
aPTT ( factor VIII is in the intrinsic pathway)
Definitive diagnosis:
identification of low factor VIII or IX thru a specific factor assay

2. DISSEMINATED INTRAVASCULAR COAGULOPATHY

Coagulation Factor deficiency 2 pathological processes taking place simultaneously:
A. Hemophilia activation of fibrinolytic system
Hemophilia A Consumption of Coagulation Factors
Hemophilia B Formation of multiple thrombi leading to consumption of coagulation
B. DIC factors.
C. APAS Inappropriate coagulation activation formation of thrombi
D. Hypercoagulable State (Thrombophilia) consumption of coagulation factors activation of fibrinolytic
system
1. HEMOPHILIA
a. HEMOPHILIA A Settings associated with DIC development:
DIC is a secondary disorder, there will always be an inciting factor
Missing or low levels of Factor VII sepsis (mostly gram negative bacteria, some gram positive and viruses)
majority of hemophilia patients (9/10 of all cases) o endotoxins; but gram positive bacteria has endotoxin-like property
Trauma and tissue destruction
b. HEMOPHILIA B Malignancy (lung Ca, Leukemia)
missing or low levels of Factor I Obstetric complication
Complications:
shock
hemorrhage preceded with wide spread thrombosis
renal failure

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Laboratory Dx: hematologic
obstetric, pulmonary
1. Fibrinogen Decreased one of the most important factor in dermatologic
the coagulation cascade cardiac
consumption coagulopathy ocular
2. PT Prolonged consumptive coagulopathy- all adrenal
factors are involved all factors are musculoskeletal
3. aPTT Prolonged decreased Laboratory characteristics:

(7.2P) CLINICAL PATHOLOGY: Platelets, Blood Coagulation and Fibrinolysis Disorders; Leukocytic Disorders
Hematologic establishment of APAS:
4. Platelet count Decreased first step in coagulation is the 1. Prolonged aPTT, Russel Viper Venom time, or Kaolin clotting time
formation of primary plug Russel Viper Venom time
5. Fibrin split Increased Activation of fibrinolytic system reagent (venom) - could induce thrombosis
products destruction of fibrin If patient has antiphospholipid antibody or anticardiolipin this would
6. Protamine sulfate Increased Sensitive test for DIC: detects fibrin interfere with thrombosis, thus RVVT is prolonged)
test monomers and early fibrin Kaolin Clotting time
Fibrin monomers and early degradation products (same principle provide a glass particle as an additional surface for contact
fibrin products with fibrin split products BUT inhibition
becomes *modification of aPTT
positive earlier) ***aPTT, Russel Biper venom Time, Kaolin Clotting time - PROLONGED
Treatment:
Heparin 2. Failure to correct the test by mixing patient plasma with normal plasma
Fresh frozen Plasma (suggesting a clotting inhibitor is present)
Suggest a mixing study, get the plasma from a normal person then
3. ANTIPHOSPHOLIPID ANTIBODY SYNDROME (APAS) mix it with the patients plasma--- determine aPTT
can occur as a primary syndrome called APAS or secondary syndrome like in SLE Coagulation deficiency: normalize (due to addition of deficient
there is the formation of antiphospholipid antibodies (anticardiolipin) factor)
high titers of circulating antibodies against anionic phospholipids (e.g. cardiolipin) APAS: it will not normalize
Clue to diagnosis: false positive syphilis test because the reagent uses cardiolipin 3. Normalization of the test with freeze-thawed platelets (contain
** phospholipid surface- where the formation of blood clot takes place phospholipids) or phospholipids
**Coagulation cascade will need a phospholipid surface for it to function optimally Test will normalize due to addition of naturally occurring
phospholipid which is needed
in vitro: antibodies interfere with coagulation assays that make use of *** Immunologic establishment of APAS:
phospholipid reagents or are phospholipid dependent (ex. aPTT). Therefore, 4. Specific assay for anticardiolipin
coagulation tests are manifested with prolonged results; multiple thrombi in any
organ system 4. HYPERCOAGULABLE STATE (THROMBOPHILIA)
in vivo: induce a hypercoagulable state Deficiency in naturally occurring anticoagulants (Factor C and S)
*** Main manifestation of patients with APAS would be multiple thrombi Factor C is Vit k dependent
formation occurring in many organ systems (hypercoagulable) -rare
** The effects of antibody in vivo interfering in the coagulation Leiden mutation (FACTOR V)
**patient with repeated abortion work up for APAS possible pregnancy -Factor V cannot be inactivated by Protein C due to the mutation
Organ systems involved:
peripheral venous system
CNS