Blood Flashcards
Sites of Hematopoiesis
Intramedullary: within the bone marrow. Normal in children and adults
Extramedullary: outside the bone marrow- the spleen and liver, normal in the fetus. Abnormal in disease states
Hematopoiesis
Fetal yolk sac at 3 wks gestation develops mesenchymal blood islands
These stem cells migrate to the liver and spleen at week 6-12
Red bone marrow develops bone at the 12th week
Red marrow stops at puberty, long bones only then generate blood
Myeloid cell line (common myeloid progenitor)
Granulocytes (N E B)
Monocytes
RBC
Platelets
Lymphoid progenitor cell line
Dendritic cell
B
T
NK
Maturation of the RBC
Polychromatophilic normoblast (Blast cell) within the bone marrow have nuclei.
The reticulocyte is the blast cell without a nuclei. It still has some ribosomes and is making hemoglobin. It is released from the bone marrow and within hours it becomes RBC.
Erythropoietin Physiology
EPO is released due to hypoxic stimulation of peritubular cells in kidney via colony stimulating factor-E (erythrocyte)
Stimulates the production of RBC via progenitor line as well as release of RBC from bone marrow.
Normal hemoglobin content of RBC cytoplasm
32-36%
Hemoglobin Biochemistry
HbA is 4 globin chains each with a heme molecule. Usually is 2 alpha 2 beta.
Heme is a protophorphyrin ring with 1 atom of Fe2+ which can bind reversibly with 1 molecule of oxygen
Storage of Dietary iron
Within the liver as Ferritin Fe3+ with apoferritin
Within the liver as hemosiderin (partially degraded ferritin)
Storage of iron in bone marrow
Liver stores are transported to the bone marrow as transferrin, which is Fe3 and apotransferrin
Normal Forms of Hemoglobin
HbA2: 2 alpha2, 2 beta2 in adults.
HbF: 2 alpha 2, 2 gamma2 in infants with some A2
RBC senescence
Driven by degradation of metabolic proteins due to the lack of a nucleus over 120 days.
Once enzymes run out the cells lose membrane fluidity, and water enters cells making them spheroid, rigid, and fragile
Extravascular Hemolysis Physiology
In splenic sinusoids: the old RBCs get stuck, and are destroyed by splenic macrophages.
Also occurs in liver.
In bone marrow, unnecessary progenitors are destroyed by BM macrophages.
This causes release of unconjugated bilirubin which gets excreted in hepatic bile.
Also occurs in spleen with IgG reaction on RBC
Intravascular Hemolysis Physiology
Fibrin clots, Immune response etc causing hemoglobinemia
Leukocyte Maturation Times
Neutrophils: 12 hours
Eo; 3.5 days
Monocyte: 3 days
Neutrophil development
Proliferation within the bone marrow consists of myeloblast, promyelocyte, myelocyte, metamyelocyte.
Metamyelocytes can only mature but not proliferate.
Metamyelocytes develop to bands and then neutrophils.
The ‘band’ is the nucleus that is not segmented. Normal is 5% of neutrophils or less in peripheral smear
Neutrophil granule contents
Myeloperioxdase
Lysozyme
Alkaline phosphatase
Eosinophil function
Antigen presenting cell
Releases major basic protein (MBP) and acid phosphatase
Releases other cytokines
Basophil function
Has Surface IgE receptors that cause allergic inflammation
Releases histamine and cytokines
Similar to mast cells
T cell physiology
Proliferation in thymus. Those that do not recognize self antigens go through apoptosis.
If T cell contacts foreign antigen from lymphatic drainage then it becomes sensitized.
May become CD4 helper T cells.
May become CD8 cytotoxic t cells
May be come regulatory T cell to suppress response
B cell physiology
lymphoid progenitor in bone marrow develops to naive B cells, which migrate to germinal centres of lymph tissue.
When exposed to antigens they transform to plasma/memory cells which produce antibodies
NK cell physiology
matures directly from common lymphocyte progenitor and has non specific immunity capable of killing tumour cells or virus infected cells without prior exposure
Control of thrombopoiesis
Thrombopoietin is produced by the kidney, liver and smooth muscle
It binds to receptors on platelets and is destroyed. The lower the number of platelets the more the hormone circulates.
Platelet plasma membrane structure
Contains negative glycocalyx carbohydrates making it very negative and repelling other platelets
Has proteins for binding collagen directly
Has proteins for binding vWF
FVIII
Cofactor that circulates bound to vWF, produced in liver
Binds with FIX
Extremely unstable unless bound to vWF.
FV
Cofactor produced by megakaryocytes, stored in alpha granules, 20% circulates in platelets
Binds to FX
vWF Functions
Carries FVIII
Binds to collagen
Binds to GpIb on platelets especially in high shear stress environments
Has a heparin binding site
ADAMTS-13
Breaks down vWF multimers
Deficiency causes familial TTP
Can be inhibited by inflammation causing immune-mediated TTP
vWF Disease, Inherited
Autosomal dominant resulting in decreased vWF and mucocutaneous bleeding. Made in endothelium and megakaryocytes
Type 1: asymptomatic, heterozygous
Type 2: Qualitative, mild symptoms that generate multimers
Type 3: Homozygous, hemorrhage
vWf Disease, acquired
Autoantibody generation against vWF, associated with aortic stenosis and VAD
Functions of thrombin
-Fibrinogen to fibrin monomers
-Activates FV,FVIII, IX
-Cross links with FXIII
-Activates platelets, thromboxane
-Endothelial stim: tPA, NO, prostacyclin
-Activates protein C, thrombomodulin
Thrombin is the final serine protease in the cascade
Platelet Granule contents
- ADP causes aggregation
- Epinephrine
- Calcium
- Thromboxane: aggregation and vasoconstriction
- FV
- vWF
Thombomodulin function
Constititutively expressed on endothelial cell surface
Acts as cofactor to the activation of protein C by thrombin
In combination with protein S, proteolytically clears FVIII and FV
Antithrombin III
Inhibits thrombin, and F 9-12 as a serine protease
Produced in the liver
Increased allosteric function of anti FIXa and FXa via minimal heparin pentasaccharide sequence
Extrinsic Clotting pathway
- Exposed TF activates FVII(Serine protease) on endothelial surface
- Happens on negative endothelial surface, with presence of positive Ca ions
- FVIIa acts on X
Intrinsic Clotting pathway
Prekallikrein, HMWK and XII activate in the presence of tissue injury (polyanions, -). Also activated via thrombin
Activates XI, IX.
This complexes with FVIIIa on platelet surface to create tenase complex (IXa+VIIIa), which cleaves X and creates common pathway
Common clotting pathway
Xa+ Va on platelet surface causes thrombin generation
Rosenthal correction factor for pH and temperature
Water increases 0.017 pH unit for every degree decrease in blood
Collection bags of Canadian blood
Buffy coat collection set: whole blood cooled then centrifuged, separating plasma, Buffy coat, and RBC. RBC then LR. Allows for pooled platelet product creation.
Whole blood collection set: cooled, LR (WBC and PLT removed) and then RBC/plasma extracted
Average CBS RBC unit
293 mL containing 56 grams of Hgb, Hct 0.68. One unit typically increases [Hgb] by 10 g/L in adults.
American= 300-400 mL, hot 55-65%, increases hct by 3%
Conditions in which blood products may be returned to inventory
Bag intact, pass visual inspection, maintained an acceptable temperature or the RBC have not been outside of fridge for more than 60 min
CBS RBC shelf life
-42 days from the time of collection
-24 hours if entered without use of sterile connection device, if stored at 1-6C or 4 hours if stored above 6C
-unit irradiated at CBS within 14 days and may be stored for another 14 days
-
CBS RBC unit transportation
1-6 C in temp controlled storage with alarm, fan and continuous monitoring.
1-10C if transport is less than 24h
Must have documentation to allow tracing of each portion of transport.
Manufacture method of CBS pooled platelets
From whole blood via Buffy coat collection method using CPD anticoagulant. Plasma top layer and RBC bottom layers removed, leaving Buffy coat containing PLT and WBC.
4 donations of same ABO group with plasma from one of the same 4 donations (usually male) are pooled then LR.
Pool is Rh neg if all donor units are Rh neg.
Produced within 28 hours of collection, stored for 7 days
Average CBS pooled platelet unit
342 mL with 300 x10^9
Manufacture method of CBS Apheresis platelets, average aperheresis platelets
Flow cytometer separates RBC/WBC from platelets and plasma. Contains 242 mL with 370 X10^9
Use of Apheresis platelets vs pooled Buffy coat platelets
Aperesis is single donor. Can be used for HLA typing when recipient has demonstrated anti-HLA antibodies for platelet refractoriness
Storage of platelets
20-24C under continuous agitation for 7 days since collection
Expires 4 hours after opening unless opened under sterile conditions for aliquot preparation, in which case the aliquots can be stored for 7 days
CBS Frozen plasma manufacture
Whole blood collection in CPD, red cell reduced by centrifugation, either Buffy coat removed via centrifugation or through filtration. CPD FP frozen within 24 hours of collection.
Not considered LR because processing removes cells but remaining plasma has variable WBC
CBS fresh frozen plasma manufacture
Apheresis generation removes plasma, collected and frozen within 8 hours and labelled as apheresis fresh frozen plasma
Difference in use between FP and FFP
AFFP contains 87% of FVIII and 0.7 IU/mL FVIII
FP contains 70-75% of the FVIII and 0.52 IU/mL
Other factors are similar. Therefore AFFP should be used with isolated FVIII or VWF deficiency if recombinant products are unavailable.
FV also reduced in FP.
1 apheresis donation=2 whole blood donations by volume
General indications for plasma transfusion
Bleeding, severe liver disease, DIC, massive transfusion, invasive procedure on warfarin before vit K can work and PCC is not available, rare protein deficiencies, TTP/HUS by plasma exchange, other indications for plasma exchange
Indications for cryosupernatant plasma transfusion
Treatment of TTP and HUS by plasma exchange, or multiple plasma deficiency especially where fibrinogen replacement is not required (eg. warfarin replacement that does not require fibrinogen)
Contraindications of plasma transfusion
- Volume replacement use alone
- single factor deficiency with available recombinant product/virally inactivated product available
- reversal of therapy with INR below 1.8
Contraindications of cryosupernantant plasma transfusion
- conditions that require fibrinogen replacement
- FVIII or VWF replacement
General adult dosing for FP/FFP transfusion
10-15 mL/kg will achieve 30% of plasma clotting factor activity. (US recommends 10-20 mL/kg)
-5-8 mL/kg will usually reverse warfarin
-should be aided by serial coagulation testing
Generally, small adult= 3 units, large=4 units
Thawing of FP, FFP, CSP
Thawed in circulating water bath of 30-37C for 30-60 min or in dry system for 12-30 min depending on volume and equipment.
Thawed product stored at 1-6C for 5 days
Administration/storage of thawed FP, FFP, CSP products
Transfuse FFP immediately or store at 1-6C for 5 days with ACD-A.
Apheresis thawed samples in sodium citrate are stored for 24 hours.
Once thawed, products cannot be refrozen
Return of plasma products
If thawed plasma needs to be returned it can be done so it should be kept in refrigerator or transport device and only if passes visual inspection and bag is intact. Must not be out of freezer for 30 min
Frozen plasma product storage
-18C or colder in a controlled monitored freezer for 1 year
Usual CBS cryoprecipitate unit
10 mL containing 285 mg of fibrinogen.
10 units usually contains 4 grams fibrinogen
Indications for cryoprecipitate transfusion
Fibrinogen replacement for acquired hypofibrinogenemia, massive transfusion.
Typically fibrinogen less than 1.0 g/L in DIC and less than 1.5 in massive transfusion and leukaemia.
No longer recommended as FVIII replacement.
Fibrinogen deficiency should be documented.
Contraindications for cryoprecipitate transfusion
Any use other than fibrinogen replacement with 1.0 or less or in massive transfusion with coagulopathy.
Should not be used to make fibrin glue, hemophilia treatment or VWD.
Calculation of fibrinogen dose
Estimated blood volume x (1-hct)= plasma volume
Desired fibrinogen-actual fibrinogen x plasma volume= Amount needed.
Amount needed/285 mg fibrinogen per unit= # units needed
Usual adult dose is 1-2 units per 10 kg body weight
10 units increases fibrinogen by 0.7-1 gram/L
Storage of cryoprecipitate
-18C or colder for a max of 12 months, must not be out of the freezer for longer than 30 min.
-Once thawed, transfuse within 4 hours
If cryoprecipitate is pooled with NS, all units will need to be used within 4 hours of opening. If not used immediately the product should be returned to the storage device and returned
Human serum albumin size
67 kDa
Oncotic activity of albumin
1 gram of albumin attracts 18 mL water. 25 g expands volume by 450 mL.
5% is iso osmotic, 25% is hyperoncotic and therefore expansionists plasma by 4-5x volume infused
Serum albumin loss in hemorrhage
12 grams per 500 mL of blood lost via hemorrhage
usual sodium concentration of albumin
130-160 mEq/L
Administration of albumin
max 5 mL/min for 5%, 1-2 ml/min for 25%. Don’t mix with hypotonic solutions like SWFI because it can lead to hemolysis. Infuse within 4 hours once opened.
Disadvantages to albumin infusion
Cost compared to crystalloid
Dilution of hemoglobin
circulatory overload
dilution of plasma proteins and coagulopathy
List of most common causes of transfusion associated mortality
- Transfusion-related acute lung injury (TRALI)
- TACO
- Hemolytic transfusion reactions (non ABO more common than ABO)
- microbial contamination
- allergic reaction
Diagnostic criteria for TRALI
- clinical diagnosis
- hypoxemia and pulmonary edema within 6 hours of transfusion
Mechanism of TRALI
HLA incompatibility between donor WBC and host immune system.
American Red Cross RBC transfusion recommendations
<6 g/dL in young healthy patient
<7g/dL in critically ill patient
<7-8 in patients with cardiovascular disease
STS recommendations for transfusion trigger on CPB
<6 g/Dl for patients on CPB with moderate hypothermia, but high risk patients need higher hemoglobin levels
Evidence based practice on RBC transfusion
liberal transfusion triggers of 9-10 have no difference in primary outcome compared to restrictive 7.5-8 transfusion trigger
Minimum FDA standard for stored RBC’s
75% remain in circulation 24 hours post transfusion. (Longer the storage time the less RBC survival due to storage lesion)
- less than 1% hemolysis
- no consideration of function of the cells themselves
RBC storage lesion
- Degredation of structure: membrane rigidity, aggrebility
- chemical: lactate, K, free hemoglobin increase.
- 2,3DPG decrease by 95% after 21 days
- depletion of NO from microvasculature from free hemoglobin constriction
RBC size
Approx 7 microns
Mechanism of free hemoglobin injury
Free hemoglobin (hemolysis, transfusion storage lesion) causes vasoconstriction in microvasculature, causing depletion of nitric oxide. Leads to insufficient NO bioavailability
Evidence for new vs old blood
No difference between stored blood for shorter and longer storage blood, but few patients receive very old blood. Very fresh blood and medium blood have similar outcomes
Usual recipe for massive transfusion, empiric
1 RBC: 1FFP: 1Plt theoretically prevents dilutional coagulopathy
General Red Cross recommendation for platelet therapy, dose of adult platelets
Give for less than 50 k/microL. Standard dose is 1 unit/10 kg body weight. 6 units from whole blood (apheresis) increases put by 30
Most common/highest cost transfusion reaction
Platelet bacterial contamination. 1 in 3000 bags are contaminated.
TRALI is most often associated with platelet transfusion, due to women not being excluded from being platelet donors and high female rates of HLA antibodies
Fibrinogen content of FP
4 units of CBS FP contain 2.5 grams fibrinogen
INR and FP/FFP transfusion
FFP fails to improve INR that is less than 1.6
INR designed to follow anticoagulant dosing with Vit K antagonists and TEG may be normal when INR less than 2
Blood administration set filter size
170-260 micron
Hensley rapid transfusion definition
1 entire blood volume replaced in 24 hours
Disadvantage to erythropoietin administration
Thrombotic events and promotion of tumour growth
Hensley definition of ANH
Blood withdrawn from the patient prior to heparinization and then read ministered after CPB
Benefit of ANH
- Reduced allogenic transfusion, especially with greater than 800 mL phlebotomy.
- low volume ANH is shown to decrease intraoperative RBC transfusion, but no outcome change
Evidence for cell salvage in cardiac surgery
Use decreases risk of transfusion by 34% and saves 1 unit PRBC transfusion per patient on average
Functions of Thrombin
Fibrinogen to Fibrin
FXI, FXIII positive feedback loop activation
Cofactor V VIII activation
Protein C pathway inhibitin V VIII
Platelet aggregation
Stimulates tPA release from endothelial cells
Platelet granule contents
Fibrinogen, vWF, Factor V, Calcium, Serotonin, Plasminogen, Platelet factor 4
FVIII Physiology
Produced primarily in hepatocytes
Extremenly unstable except when bound to vWF
Binds with FIX and vWF in coagulation complex
Activation of Intrinsic pathway (Contact activation)
FXII binds to negatively charged surface (polyanion either from surface or platelet secretion).
FXIIa activates prekallikrein to stimulate fibrinolysis and inflammation, and FXI to intrinsic pathway.
Activation of extrinsic coagulation pathway
TF in sub endothelial system activates is presented via injury to its ligand FVII
3 Major coagulation cascade complexes
- TF +FVIIa
- IXa +FVIII (Tenase)
- Xa+ Va (Prothrombinase)
All complexes require Ca, anionic phospholipid surface,, cofactors
Action of heparin
UFH binds to ATIII AND THROMBIN and forms a complex: the complex inactivates thrombin, 9,10,11,12
Short heparin binds to ATIII ONLY but the complex mainly inactivates FXa.
LMWH binds less to proteins so is more predicable.
1 USP unit of heparin
keeps 1 mL citrated re-calcified sheep blood liquid for 1 hour
Heparin pharmacokinetics
Half life:30 min at small doses, 2-3 hours at large doses. Half life is dose dependent.
Onset 1 minute.
Renal elimination or by endothelial cells
Heparin chemistry
glycosaminoglycan, negatively charged, very acidic
Hensley Heparin prime content
CPB prime should contain heparin at the same concentration as that of the patient (3-4 units/mL) to account for the expanded plasma blood volume
ACT and temperature
At temperatures below 25, act is so prolonged that alternative tests should be considered
Hensley ACT minimum recommendation
400 s
Aprotinin and ACT
Aprotinin prolongs the ACT in cases where celite activators are used, therefore consider titrating heparin to ACT of 750 with celite. Kaolin is not affected by aprotinin
Clinical use of heparin concentration testing
In hypothermia under 25C
To maintain heparin concentrations of 3-4 u/mL which may suppress thrombin more effectively.
Can be used in context of aprotinin
Must be used with ACT as variable sensitivity to heparin between patients is known
Can determine protamine dose, only if blood volume is constant
Initial Loading Dose of Heparin
300-400 units/kg, usually to a max of 35-40 thousand units as patient weight tends to max at lean body mass, with maximum lean body mass usually 90-110 kg
Usual acceptable ACT/heparin dose for off pump revascularization procedures
200-300 seconds with dose of 100-300 units/kg
Use of heparin concentration monitoring on CPB
whole blood concentration targeted for 3-4 u/mL are sufficient.
Use of [Heparin] substantially increases the amount of heparin given but suppresses thrombin formation, at the expense of heparin rebound and platelet activation
Patient sensitivity to heparin varies, therefore another clotting test (simultaneous ACT) is recommended
LMWH vs UFH for CPB
LMWH does not bind to thrombin as well (primarily anti Xa activity) compared to long chain UFH. Thrombin inactivation is pivotal for CPB
LMWH has a long half life
LMWH poorly neutralized by protamine
Factors decreasing the response to heparin
- ATIII Deficiency (familial or acquired)
- Hypercoaguable states (Sepsis, arteriosclerosis, pregnancy, HIT, thrombocytosis)
- Nitroglycerine
- Drug protein binding (acid glycoproteins, immunoglobulin)
- Extremes of age
Use of ATIII supplementation
For suspected heparin resistance, if more than 600 units/kg given.
2 units of FFP=500 units of ATIII
Clinical approach to heparin resistance
- Add more heparin, though usually >4u/kg does not tend to improve anticoagulation
- Add 2 units or 500 mL FFP
- Add ATIII concentrate
- Accept the ACT due to the wide acceptability of range for safe bypass
Serum vs plasma
Serum is obtained after the sample is clotted. Contains no fibrinogen. Contains no cells.
Plasma has clotting factors (including fibrinogen.) Used for testing coagulation
Polychromasia
Increased reticulocyte count; reticulocytes released indicate early release of immature RBC
Hypochromasia
Less Hgb inside the cell, usually due to insufficient hemoglobin production.
MCV size interpretation
Large cells: insufficient cell division usually caused by vitamin deficiency. Target cells have large cell membranes caused by liver failure.
Small cells: cell division is normal but don’t contain enough Hgb, usually thalassemia or iron deficiency
Prothrombin time testing method
Adds thromboplastin (tissue factor and phospholipid) and calcium. Simulates extrinsic pathway via FVII activation and common pathway activation. Most affected by Coumadin, as FVII gets affected first by VitK block. Done with centrifuged, platelet poor plasma
activated partial thromboplastin time testing method
2 reagents are added separately: thromboplastin (TF and phospholipid), calcium, and negatively charged activator like kaolin. Done with centrifuged, platelet poor plasma
Beta Thalassemia pathophysiology and management
- reduced/absent production of beta chain in hemoglobin, leading to excess alpha chain production and precipitation. This causes extracellular hemolysis in spleen, and increased compensatory iron
- Tx: transfusion, iron chelation and splenectomy
Heparin Induced Thrombocytopenia Pathophysiology.
Most often occurs 5 days following continuous heparin, average is 9 days. Antibodies are created between heparin and PF4 complex, causing aggregation of platelets and thrombocytopenia.
Safe time for heparin administration following HIT
Antibodies usually disappear 50-85 days following the most recent exposure to heparin
Bivalirudin use
For CPB is 1 mg/kg bolus then 2.5 mg/kg/hr for CPB. While it has a shorter half life than heparin, at 24 min, there is no reversal. Direct thrombin inhibitor
Usual protamine dosing
1 mg of protamine neutralizes 100 IU of heparin. Most clinicians give 0.6 mg/100 IU to 1:1 protamine.
ACT post protamine
Should be no more than 10% above the value before heparin administration
Protamine administration speed
Greater than 3 minutes at 3 mg/kg. Faster than that causes dropping of SVR and PVR which can then be partially resolved with addition of volume. 10 minutes recommended.
Protamine PVR hypertension reaction cause
Pulmonary vasoconstriction thought to be due to heparin-protamine complexes stimulating pulmonary macrophages to produce thromboxane
Treatment of adverse protamine reactions
- Usually low BP within 10 min of protamine is because of protamine, but consider LV failure or hypovolemia.
- If hypotension and normal/low SVR, treat as anaphylactoid. Give fluid and epi, consider steroids
- if low BP and high PAP consider isoproterenol and milronone. Occasionally, additional heparin will resolve the reaction
Blood shear stress equation, definition
Force applied to an area of liquid confined between 2 plates that is sufficient to set the liquid in motion.
Stress= viscosity x shear rate
Where; shear rate is the velocity relative to the plates
Blood viscosity relationship
Poiseuille: flow is related to r^4 and inversely related to viscosity. As diameter decreases the shear rate decreases.
Flow is lowest and viscosity highest in postcapillary venules
Hematocrit and viscocity
Is a linear relationship
Blood temperature and viscocity
Not linear. Temp decrease of 10C increases viscocity. 20-25%. Highest viscocity is seen at temperatures below 25C
Perfusion pressure and blood flow relationship
Per Poseuille, perfusion pressure cannot be an adequate marker of flow because pressure depends on flow and viscocity, and viscocity depends on temperature.
Optimum hematocrit for o2 delivery (Gravlee)
30%. Lower amounts have too little hemoglobin, too high causes an increase in SVR due to increased viscocity
Recommended max cell saver suction
Less than 150 mmHg
Ratio of blood to anticoagulation in cell saver
1-5 to 1:10 with a target of 15 mL/100 mL blood with heparin or 70
