Heme Lecture Final

Heme

Blood

Stasis

to halt

What are the goals of successful hemostasis? (4)

- maintain blood flow
- form a barrier
- limit the barrier
- dissolve the clot

What are the steps of coagulation?

1. vasoconstriction
2. temporary platelet plug
3. coagulation
4. fibrinolysis
5. regeneration

Primary hemostasis

temporary platelet plug

secondary hemostasis

coagulation

what are the 3 elements of hemostasis?

- vasculature
- platelets
- proteins (plasma factors)

coagulation cascade definition

sequence of enzymatic reactions leading to the formation of fibrin

Fibrinolysis

- breakdown clot

- elements: proteins

- results: prevent excess clotting

Hemophilia

- excessive bleeding
- primary or secondary malfunction

Thrombophilia

- excessive clotting
- fibrinolysis malfunction

Quality Assurance (QA)

coordinated efforts of all departments to organize all activities in the lab to provide the best service to pts and providers

Quality Control (QC)

the process of monitoring specific testing systems

Common lab associated agencies

- JCAHO (joint commission)
- CAP (college of american pathologists)
- COLA (commission on office lab accreditation)

Accuracy

the exactness of a measurement in comparison with true value

Precision

closeness of values to each other (reproducibility)

Reliability

ability to maintain both accuracy and precision over a period of time

Delta Checks

comparing the result of an analysis of pt sample with previous results
goals to ID:
1. change in pt condition
2. sample issues

Reference range

normal range of a healthy population of similar demographics (age/sex/race/location)

Standards

solutions with a single known conc. used to make standard curves

Primary standard

reference material that is of fixed and known composition (pure form)

Secondary standard

concentration is obtained by comparing against primary standard

Diagnostic Sensitivity

proportion of pts WITH a disease showing a POSITIVE result

Sensitivity formula

TP/(TP+FN)

Diagnostic Specificity

proportion of pts W/OUT a disease showing a NEGATIVE result

Specificity formula

TN/(TN+FP)

Systematic Errors

- errors in test system or method

- constant: magnitude of error remains constant throughout the range

- proportional: magnitude of error inc w/ the conc. of the substance being measured

Random Errors

- occur without prediction or regularity
- less likely to happen in repeated pattern

Calibrators

standards used to define a standard curve (not required every day)

Controls

used for QC to confirm system is working (must be performed everyday before pt testing)

Shift

a sudden shift of values

Trend

a gradual continuous shift of values in one direction over the span of 6 or more values

1(2s) Rule

control value outside of 2SD (random)

1(3s) rule

a control value outside of 3SD (random)

2(2s) rule

2 consecutive control values outside of 2SD (systematic)

R4s rule

2 consecutive values are > 4SD apart (random)

4(1s)

4 consecutive values are outside of 1SD on the same side (systematic)

10x

10 consecutive control values fall on the same side of the mean (systematic)

External quality control

outside agency sends the controls for calibration (required)

Platelets

disk-shaped cells necessary for primary hemostasis

Where are platelets formed?

megakaryocytes in the bone marrow

normal reference range for platelets

150-450 x10^3 /uL

Where are platelets found?

2/3 circulating blood
1/3 spleen

average lifespan of platelets

8-12 days

4 regions of platelet

1. peripheral zone
2. structural zone
3. organelle zone
4. membrane systems

Peripheral zone

adhesion and aggregation (glycocalyx)

glycocalyx

outer membrane surface rich in glycoproteins which contributes to the negative charge of platelet surface

glycoproteins

Ib/IX: receptors for vWF, thrombin, ristocetin
IIB/IIIa: fibrinogen, vWF
Va: thrombin

Structural zone

structure and support (microtubules and microfilaments)

microtubules

made of tubulin and support the normal disc shape

microfilaments

contain actin, responsible for shape change

Organelle zone

storage and secretion
1) Alpha (protein) and Dense (non-protein) granules
2) mitochondria: formation of ATP
3) glycogen: food for the platelet
4) lysosomes: proteolytic enzymes

membrane systems

storage and secretion
OCS: direct communication btwn intra/extracellular compartments
DTS: calcium regulation

Platelet function

1. maintain blood vessel integrity
2. primary hemostatic plug
3. secondary hemostatic plug
4. aid in healing injured tissue

How doe platelets maintain vascular integrity?

- requires contact btwn platelets and endothelium
- release cytokines and growth factors
- block gaps in lining

What are the stages of primary hemostatic plug?

- adhesion
- activation
- shape change
- secretion
- aggregation

Adhesion

Tissue injury: exposure of vWF and collagen
platelet sticks to vWF/collagen via glycoprotein Ib/IX

Activation

injury and adhesion trigger release of platelet agonists (bind platelet surface receptors)

Shape change

- carried out by microfilaments
- inc surface area
- brings granules closer to OCS for secretion

Secretion

granules discharged after fusion of OCS to granule membrane (requires ATP)
- positive feedback loop leads to aggregation

Aggregation

fibrinogen binds to GP IIb/IIIa on activated platelet
- Ca dependent
- primary phase: loose, reversible

secondary phase of aggregation

platelets begin to release ADP and TXA, irreversible
- aspirin inhibits COX

Secondary hemostatic plug

- fibrin reinforced
- coagulation cascade leads to production of thrombin
- thrombin transforms fibrinogen to fibrin

Aid in healing injured tissue

- clot retraction
- size of clot dec over time
- edges of endothelium slowly grow closer

Platelet aggregate growth limiting factors:

1. blood flow (wash away promoting factors)
2. prostaglandin 12 (aggregation inhibitor)
3. dense-granule substances cause inflammation and vasodilation (inc blood flow)

What hormone regulates development of platelets?

TPO (thrombopoietin)

Where do they develop (microenvironment)?

stromal cells

Megakaryoblasts

- 6- 24 um
- basophilic staining
- no granules
- no lobes
- high N:C ratio

Promegakaryocyte

- 14-30 um
- basophilic (some pink)staining
- few granules
- bilobed
- moderate N:C ratio

granular megakaryocyte

- 25-50 um
- majority acidophilic (pink)
- extensive granules
- many lobes
- moderate N:C ratio

Mature megakaryocyte

- 40-100 um
- acidophilic
- extensive granules (demarcation zones)
- many lobes
- low N:C ratio

How many megakaryocytes are in normal marrow?

15 million

Thrombopoietin (TPO)

major humoral factor regulating megakaryocyte and platelet development

As TPO decreases:

platelet count increases

Normal platelet reference range

150,000-450,000

Platelet aggregation study

Measure how long it takes for a clot to form by adding reagents to plasma (light transmittance increases with aggregation)

Platelet function assay

measures platelet adhesion and aggregation

Normal CT on COL/EPI test

normal platelet function

prolonged COL/EPI normal COL/ADP

aspirin-induced platelet dysfunction

prolonged COL/EPI prolonged COL/ADP

-Von Willebrand's disease
-thrombocytopenia
-platelet defect other than aspirin

Intrinsic pathway

-initiating rxns (contact activation)
-involves: PK, HMWK, XII, XI, IX, and VIII
-requires enzymes/protein cofactors, contact w/ negatively charged surface

What is the lab test for the intrinsic pathway?

APTT (activated partial thromboplastin time)

Extrinsic pathway

-factor VII
-tissue factor initiator
-glycoprotein exist of non-vascular cell surface
-TF exposed to plasma when vessel is injured

What activates FX leading to the common pathway?

FVIIa/TF complex

What is the laboratory test for extrinsic pathway?

PT

Common pathway

-activation of factor X
-involves: FX, FV, FII, FI, FXIII
-intrinsic FX activated by FIXa
-extrinsic FX activated by VIIa/TF w/ Ca cofactor

Factor XII (hageman)

-activated to XIIa by negatively charged surface (damage/subendothelial tissue)
-no Ca or enzyme required to activate

Factor XII results in what 3 products?

1. XIIa cleaves prekallikrein to produce kallikrein
2. XIIa cleaves XI to XIa
3. XIIa cleaves plasminogen to plasmin

Factor XI

-activates IX
-requires Ca

Factor IX

-forms a complex w/ platelet phospholipid membrane surface and FVIII in the presence of Ca
-activates X to start common pathway

Factor VIII

-cofactor in activation of X by IXa
-circulates in complex w/ vWF
-activated by thrombin
-inactivated by protein C

An inherited deficiency of VII is what ?

Hemophilia-A

VWF

-made by endothelial cells, megakaryocytes, and platelets
-made as a monomer but circulates as polymers

What are the 2 functions of vWF?

-induce platelet adhesion
-bind to VIII to prolong half-life

An inherited deficiency of vWF is what?

von Willebrand's disease (vWD)

TF-factor III

-cofactor w/ FVII
-requires Ca
-activates X

Factor VII

-cofactor w/ TF
-requires Ca
-activates X
-factor w/ shortest half-life

Factor X

-converts prothrombin to thrombin
-combines w/ phospholipid membrane, Va and Ca forms Thrombomodulin complex

Factor V (proacellerin)

-converts prothrombin to thrombin
- combines w/ phospholipid membrane, Xa, and Ca forms Thrombomodulin complex

Factor II (thrombin)

-converts fibrinogen to fibrin
-activates FXIII, platelet agg, FV, FVIII, protein C, and VII (weakly)