Lesson 3: technical hemostasis

clinical symptoms of bleeding disorders?

• unusual bleeding

• unusual bruising

what conditions are common for platelet dysfunction?

drug use, uremia

thrombocytopenia

bleeding due to a quantitative platelet inadequacy most common cause

thrombosis

increased number of platelets

what sample is used for a smear evaluation?

usually collected in EDTA lavender top

• but can cause satelite around WBC

what happens if sateliting occurs in a blood smear?

• reject sample (will be falsely low)

• recollect in sodium citrate (light blue)

• platelet correction due to liquid additive

what are smear evaluations used for?

examines: 

• distribution 

• clumps/sateliting 

• size

• morphology

• obtaining an estimate count

what is the normal size for platelets?

2-5 micrometers

what indicates that immature forms are being released early?

seeing strands in the smear

principle of in vivo (template) bleeding time

measure the interval required for cessation of bleeding following a skin puncture on the forearm (2 cuts 1mm deep, 5mm in length with pressure cuff set to 40mmHg)

reference range for bleeding time

1-9 mins

why is bleeding time not widely used anymore?

• not specific or sensitive

• many false positives

characteristics of PFA-100

• screening test for symptomatic patients 

• detects platelet function abnormalities 

• better than in vivo BT and cheaper than platelet aggregation studies 

what sample is used for PFA -100?

whole blood collected in light blue tube

• placed in CEPI and CADP cartridges 

which cartridge is mainly used in PFA-100?

CEPI (collagen and epinephrine)

principle of PFA-100

1. vaccuum pulls blood sample from cartridge reservoir through capillary and exposes it to a collagen-coated membrane (induces platelet adhesion)

2. agonist (ADP or EPI) causes platelet activation (release of granular contents)

3. clot forms at aperature 

4. reports closure time 

reference range for PFA-100

CEPI = less than 146 seconds

CADP = less than 116 seconds

what does a PFA-100 test indicate if CEPI is abnormal but CADP is normal?

most likely aspirin interference 

what does a PFA-100 test indicate if both CEPI and CADP are abnormal?

platelet dysfunction 

principle of platelet aggregation studies

• has PPP and PRP samples tested with an agregometer

• an agonist is mixed with the PRP sample at room temp one at a time 

• platelets will aggregate and the more they do, the more they fall, and an increase in %T is seen 

Samplesfor platelet aggregation studies

whole blood collected in light blue top

less than three hours old, rested for 30 mins

• PPP=platelet poor plasma is the blank (less than 10×10³/microliters)

• PRP=platelet rich plasma is test (200-300×10³/microliters)

if the PRP is out of the range, what do you do?

dilute it with the PPP

what agonists are used in platelet aggregation studies?

• ADP

• EPI

• collagen

• arachidonic acid

• ristocetin 

• thrombin

reference range for platelet aggregation

any variation from normal indicates platelet function disorder 

whole blood aggregometry

• detection of aggregation with impedance (change in current)

• platelets collect on electrodes 

• more aggregation = more current

• no PRP prep needed 

principles of lumiaggregometry

• instument is a “chrono-log”

• measures platelet aggregation and secretion 

• detects secretions with chemiluminescence

• reagent=firefly luciferin 

• reacts with ATP released from dense bodies 

• aggreagtion measrued with various agonists (start with thrombin)

platelet evaluation

• screening

• start with platelet cont 

• automated (in EDTA tube)

• count based on size 

• cheap and easy

• acanthocytes and schistocytes can be falsely included 

• mean platelet volume

MPV

(platelet number x 10)/RBC count 

reference range for MPV

6.8-10.2 fL

screening for Heparin inducted thrombocytopenia

serotonin release test

immunoassay for PF4

test for thrombosis

Immunoassay for B-TG

reference range for platelet count

150-440 × 10³/microliter

critical values for platelets

less than 20 × 10³/microliter

greater than 1000 × 10³/microliter

which platelet aggregation tests are abnormal for von Willebrand disease (VWB)?

(VWB) ristocetin

normal=collagen and ADP

which platelet aggregation tests are abnormal for Bernard-Soulier syndrome (BSS)?

(BSS) ristocetin

normal =collagen and ADP

which platelet aggregation tests are abnormal for Thrombasthenia (TSA)?

(TSA) collagen and ADP

normal = ristocetin

which platelet aggreagtion tests are abnormal for storage pool disease (SPD)?

(SPD) collagen and ADP

normal = ristocetin

which platelet aggregation tests are abnormal for aspririne-like disorders (ASA)?

(ASA) collagen and ADP

normal =ristocetin