Hematology Instrumentation SOLO 2

coagulation

• the process by which liquid blood changes into a semi-solid gel-like structure

• prevents blood from escaping the vessel lumen

Fibrinolysis

• the process by which enzymes in the blood stream consolidate or dissolve unnecessary clots

• keeps the blood flowing in a liquid state

Thrombosis

• the action of inappropriate clot formation that are no longer confined to the site of injury and may pose a risk of vessel occlusion

• deep vein thrombosis, pulmonary embolism…

Hemorrhage

• the inability of the body to sufficiently form clots and plug damage to vessels which can potentially lead to severe blood loss

• excessive blood loss, hemorrhagic shock…

hemostasis

• the process ofclot formation that arrests blood loss from injured vessels while maintaining blood flow in a liquid state

• balanced through coagulation (Pro-clot) and fibrinolysis (anti-clot)

In a normal person, hemostasis is maintained in a ….

neutral state

What can disrupt balance of hemostasis and cause a shift in either direction?

trauma, medication or other abnormal events

What are the three distinct stages of hemostasis?

• vascular reaction

• primary hemostasis

• secondary hemostasis

Vascular reaction

• Vasoconstriction

  • the action of the damaged blood vessel in order to reduce blood flow through the affected area

  • improves the platelets ability to adhere to damaged surfaces

  • achieved through smooth muscle, nerve damage, and damage of the endothelial cells which release endothelin

formation of platelet plug

• mediated by the coorperative action of the damaged blood vessel and platelets

• healthy endothelial cells promote an anti-clotting environment and are non-reactive towards platelets

• damaged endothelial have exposed areas (collagen) and release compounds (tissue factor and Von Willebrand factor) that encourage platelets to adhere and activate

formation of platelet plug steps

• platelet adhesion

  • initial contact mediated by von Willebrand factor and glycoprotein 1b

• platelet activation

  • platelet stimulation through a variety of chemicals which can induce granule release which activated nearby platelets

• platelet aggregation

  • platelet shape change due to activation causes cross-linking between platelets with the aid of fibrinogen mmolecules and GPIIb/IIIa

Stabilization of the platelet plug

• conversion of fibrinogen into an insoluble fibrin clot

• made possible by the coagulation cascade

• at the same time, platelets are releasing platelet derived growth factor which promotes regeneration of the blood vessel walls

Extrinsic pathway

• activated by tissue factor

• factor VII

• measured by PT/INR

• affected by coumadin/warfarin

Intrinsic pathway

• activated by negatively-charged surfaces

• factors XII, XI, IX, VIII

• measured by aPTT

• affected by heparin

common pathway

• merging point of both pathways

• factors X, V, II, I

• measured by both PT/INR and aPTT

Testing on the ACL TOP 750

• coagulometric/turbidimetric

  • PT/INR

  • aPTT

  • Q.F.A

• chromogenic assay

  • Anti-Xa

  • AT III

• immunoturbidity

  • D-dimer

coagulometric (turbidimetric) clot detection

• used to measure and record the amount of time required for a plasma specimen to clot

• assesses coagulometric endpoint by measuring change in optical clot density

• clot detection is based on the principle that light passing through a medium in which fibrinogen is converted to fibrin is absorbed by the fibrin strands

• light transmittance through the sample continuously decreases and is measured by the photodetector

Chromogenic assay

• direct chromogenic tests: the analyte of interest acts directly on a specific chromogenic substrate

• indirect chromogenic tests: residual enzyme activity is measured using a specific synthetic substrate with a fixed quantity of enzyme to form inactive complexes

• the reaction is measured at 405nm by continuous release of paranitroanaline from a synthetic substrate

  • light passes through the cuvette is read by an optical sensor

  • light absorbed by the solution in the cuvette

  • amount of light reaching the photodetector is converted to an electrical signal and proportional to the enzyme activity

Immunoturbidity assay

• assess the physical concentration of the analyte by measuring change in optical density

• measured the reduction of light transmittance due to agglutination of Antigen-Antibody complexes

• light complexes are inversely proportional to the D-Dimer

Analyzer components ACL TOP

Overfilled specimens will not contain enough coagulant and will have

falsely shortened PT/aPTT

Under-filled specimens will have excess coagulant and will have

falsely prolonged PT/aPTT

ACL TOP specimen requirements

• must be ran within 4 hours of collection

• aliquotted specimen plasma into false bottom can be stored for 1 month at -20C and 6 months ar -70C

• hemolyzed or clotted specimens must be recollected

ACL TOP Pre-analytical flags

• Hemolysis, Icterus, Lipemia

• tube fill height check

• clog detection

PT/INR reagents

• RecombiPlasTin2G

• diluent

aPTT reagent

• SynthASil (ss)

• calcium chloride

Fibrinogen reagent

QFA thrombin (bovine)

D-Dimer HS 500 regents

• latex reagent

• reaction buffer

Liquid Anti-XA reagent

• chromogenic substrate

• FXa reagent

Special testing reagents ACL TOPS

• Liquid antithrombin (AT III)

  • chromogenic substrate

  • FXa reagent

• Factor Assay

  • factor deficient plasma

  • RecombiPlasTin 2G/SynthASil and CaCl2

  • factor diluent

PT/INR test

• screens the extrinsic/common pathways

• factors VII, X, V, II, I

• monitors coumadin/warfarin therapy

aPTT test

• screens the intrinsic/common pathways

• factors XII, XI, IX, VIII, X, V, II I

• monitors heparin therapy

fibrinogen test

• quantitative measurement

• necessary to form clots

• help diagnose DIC, cardiovascular disease

D-Dimer test

• measurement of fibrin split/degradation production

• indicator of clot formation/breakdown

• high D-dimer can indicate higher risk of DVT, PE, or DIC occurring

Anti-Xa test

• measures the concentration of heparin in patient’s sytem

• more accurate than aPTT for monitoring heparin

ATIII test

measures patient’s own amount of anti-thrombin and therefore their ability to neutralize thrombin

Factor assays

• quantitative determination of factor concentration

• determine if patient has factor deficiency or inhibitor

QC performed during each shift on ACL TOP

• normal 1

• abnormal 3

• fibrinogen

• UFH

• LMWH

• D-dimer HS 500

Daily maintenace for ACL TOP

• enhanced clean for all probes

• replace factor diluent

• replace diluted clean B

Erythrocyte sedimentation rate

• the rate at which RBCs sediment in an hour, measured in mm/hr

• a non-specific measurment of the amount of inflammation

Inflammation affect on ESR

• acute phase reactants are realsed into the blood stream and disrupt normal negative charge

  • fibrinogen, CRP, C3/C4, haptoglobin

• gives the RBCs the ability to stack and cause them to settle faster due to increased weight (rouleaux)

conditions that increase the ESR

• inflammation

• preganancy

• anemia

• autoimmune disorders

• infections/sepsis

• some cancers

conditions that decrease the ESR

• polycythemia

• sickle cell anemia

• leukemia

• liver disease

• congestive heart failure

• inappropriate specimen (clotted/bubbles)

Traditional method ESR

• Westergren method

• involved filling long narrow tubes with citrated blood and waiting 1 hour to read level of RBCs compared to palsma

iSED principle

• utilizes quantimetric photometry to capture the moment of initial RBC rouleaux formation and calculate intensity of RBC aggregation

• small sample is injected into a microcell and then is monitored for aggregation using changes in light transmittance

iSED specimen requirements

• collected in EDTA tubes

• minimum testing volume is 100 uL

• must perform within 4 hours of collection

• clotted specimens not acceptable

Platelet Function Analyzer principles

• performs a qualitative test on patient’s blood sample (platelets)

• screens for patient’s ability to perform primary hemostasis (Plt plug formation)

• specially designed cartridges are used in conjunction with the PFA to create an in vitro model of a damaged blood vessel

PFA abnormal results can be caused by…

• acquired conditions

  • uremia

  • alcohol abuse

• inherited dysfunctions

  • von Willebrand disease

  • Bernard-Soulier

  • Glanzmann Thrombasthenia

• induced deficiency

  • aspirin

  • plavix

PFA cartridges

• consists of a collagen-coated membrane infused with a platelet agonist that initiates platelet aggregation and activation

• collagen epinephrine ran initially

• collagen ADP ran is COL/EPI gives abnormal result (>179 s), which rules out the possibility of ASA-containing drugs

PFA specimen requirements

• whole blood specimens insodium citrate tubes (NOT spun down)

• must be ran within 4 hours of collection, but at least 10 minutes after collection

• must be hand delivered to the lab

• minimum volume: 800 uL

• maximum volume: 900uL

PFA abnormal result

>179 sec

What need to be obtained if you must run a COL/ADP cartridge on the PFA?

patient’s current hematocrit and platelet count

Normal patient PFA results

• COL/EPI- normal

• COL/ADP- normal

Patient on aspirin PFA results

• COL/EPI- abnormal

• COL/ADP- normal

What disease states can result in an abnormal COL/EPI and COL/ADP

• von Willebrand disease

• Glanzmann thrombasthenia

PFA pre-analytical errors

• microthrombi/mini clots (abnormally high results/flow obstruction_

• hemolysis and lipemia (false increase)

PFA error messages

• maximum test time exceeded

  • possible vacuum leak

  • abnormal patient

• test terminated due to air leak

  • no sample added to cartridge

  • air bubbles

• test terminated due to flow obstruction

  • microthrombi

• test terminated due to insufficient sample

  • low hematocrit

  • low platelet count

• test terminated due to maximum syringe travel

  • possible vacuum leak

  • low hematocrit

Thromboelastograph Principles

• allows us to monitor the kinetic changes of patient homeostasis as their sample clots, retracts and/or lyses

• end results help determine patient’s potential to perform hemostasis

• clot’s rate, strength and stability determine if the patient ha hypo-, hyper-, or normal coagulation processes

In what two ways is the TEG analyzer used?

• thromboelastograph

  • surgical/trauma patients to determine current homeostatic ability and what products to transfuse

• platelet mapping

  • non-surgical patients that have received platelet inhibiting drugs and need their platelet function assessed

TEG specific principle

• monitors the harmonic motion of a pendant drop of blood inresponse to external vibration

• modulus of elasticity and resonant frequency increase during clotting

• analyzer measures variations in resonant frequency during clotting and lysis

TEG testing requirements

• testing must be started within 2 hours of blood collection for TEG Global hemostasis with lysis and platelet mapping

• Testing must be started within 4 hours for TEG global hemostasis

• citrated whole blood for TEG

• Heparinized whole blood for platelet mapping

TEG clot parameters

• R- reaction time, amount of time between teh start of the test and the beginning of coagulation

• Angle- the speed of clot strengthening due to the rapidity of fibrin build-up and cross-linking

• K- speed of formation of the clot from R time to a specific clot strength

• MA- maximum amplitude, the ultimate strength of the clot

• LY30- percent lysis 30 minutes after MA is finalized, based on the reduction of the tracing area that occurs between the time that MA is measured until 30 minutes after MA is finalized

• FLEV- calculates the value for the functional fibrinogen level from the MA parameter for Functional Fibrinogen tests

R is affected by …..

• coagulation factor availability

• prolonged may be resolved by FFP infusion

Angle, K, CFF-MA, and FLEV are affected by…

• fibrinogen levels

• small angle/prolonged K resolved by cryo infusion

MA is affected by….

• platelet count

• narrow resolved by platelet infusion

LY30 is affected by…

• t-PA

• high may be resolved by anti-fibrinolytic (TXA) infusion

Clot parameters curves

VerifyNow principle

• anti-platelt therapy works to inhibit platelet activation and reduce the chance of any inappropriate clot formation

• able to analyze whole blood and determine the ability of the drug to inhibit platelet activity

• uses fibrinogen-coated beads in order to quantitate the patient’s therapeutic rnage

• result is calculated base on the amount of light transmitted

• platelets will be encouraged to adhere to the beads and aggregate with nearby platelets (higher transmittance)

• patients that are on plavix will have inhibited that will be discouraged from forming aggregates with the beads (lower transmittance)

TXA2 receptor

• signals the platelet to produce TXA2 from thr Arachidonic Acid via the COX-1 enzyme

• TXA2 is a platelet activator and a vasoconstrictor

• inhibited by aspirin

P2Y12 receptor

• releases ADP stored in platelet granules

• ADP is a platelet activator

• inhibited by plavix (clopidogrel)

GPIIb/IIIa receptor

• involved with platelet aggregation with the help of fibrinogen

• inhibited by integrilin

Verify now Specimen requirements

• requires a special partial-fill sodium citrate tube

• tubes have white inner cap

Verify now reagents

PRU Test device which is an all-in-one test cartridge

Free response Q (describe PFA)

The platelet function analyzer performs a qualitative test on a patient’s blood sample, specifically the platelets. It screens for the ability to perform primary hemostasis/platelet plug formation. Specially designed cartridges are used in conjunction with the PFA to create an in vitro model of a damaged blood vessel. Abnormal results can be caused by acquired conditions, inherited dysfunctions, or induced deficiency from drugs. It uses cartridges consisting of a collagen coated membrane infused with a platelet agonist that initiates platelet aggregation and activation. A COL/EPI cartridge is ran initially and if it comes back abnorml/>179s, then a COL/ADP cartridge is run. A trigger solution is also used as a wetting agent that rehydrates the membrane for testing. It requires whole blood from a sodium citrate tube.