17, 18 - Investigation of Bleeding Disorder, Approach to Bleeding Disorder

What color tube is EDTA stored in, and what is EDTA and how does it work?

• Purple color

• It is an anticoagulant that binds Ca2+. Ca2+ is needed in many steps of the coagulation cascade, so it stops any fibrin from forming

How can these cause falsely low platelet numbers?

• Clumping

• Clot

• Inadequate mixing

• EDTA induced pseudothrombocytopenia

• Giant platelets

• Clumping causes the reader to think that it’s one large non-platelet cell

• A clot means that platelets are consumed in the fibrin mix, so platelets in that mix aren’t counted

• Similar to clotting, if we don’t mix the EDTA well, some parts of the blood will still clot and the platelets will get used up

• Patients have certain antibodies that cause clumping once platelets meet EDTA, which reduces platelet count on the reader

• Giant platelets may be read as a non-platelet

How can these cause falsely high platelet numbers?

• Microcytic RBCs

• Schistocytes

• Red cell inclusions

• White cell fragments

These are all so small that they fit into the platelet size windows which can add to the platelet count

20WBCT stands for and is?

20 minute whole blood clotting test where a dry, empty tube is filled with blood and left for 20 minutes. It should clot, but it it remains very liquidy, that means that clotting is consumed. This test is usually used to assess venom-induced coagulopathy

What color tube is 3.2% sodium citrate stored in, and what is 3.2% sodium citrate and how does it work?

It is in a blue tube, and it’s a anticoagulant that chelates calcium and is used for coagulation studies(PT and PTT). The key is that it’s reversible, so it stops coagulation until we add more calcium to restart the cascade and time it

Explain these situations and their significance in terms of performing a coagulation cascade test

• Hct>55%

• IV heparin

• Too much Hct means there’s too many RBCs and too little plasma(which is where the coagulation factors are), so the sodium citrate will bind the Ca2+ easily, leaving left over citrate which will bind the Ca2+ reagent we add to time the test, meaning that there will be less available Ca2+ to perform the cascade and prolong coagulation time

• Heparin is an anticoagulant that can prolong coagulation time, meaning that it can appear prolonged

Factors involved in coagulation cascade

1. Intrinsic pathway: 12, 11, 9, 8 into 10, 5, 2, 1

2. Extrinsic pathway: 7 into 10, 5, 2, 1

What are APTT and PT, and what do they measure?

Activated prothrombin time measures how long the intrinsic pathway takes while prolonged prothrombin time measures how long the extrinsic pathway takes

Which of APTT and PT is usually prolonged, and why?

Prothrombin time, because factor 7(The initiator of the extrinsic pathway) has a shorter half-life than all the factors, so if there’s an issue with production of factor 7, it’ll show up very fast

How does vitamin K/warfarin impact coagulation, and how does that show up in lab?

• It inhibits the factors that require vitamin K. One of those factors is favor VII, so it’ll start with PT initially being more prolonged while APTT is still normal, but if it continues, then both will become prolonged

If APTT only is too long, what can the causes be and how can we tell the causes apart?

1. Factor deficiency: A deficiency of one of the factors of the intrinsic pathway

2. Factor inhibitor: The factor amount is normal, but something is inhibiting it

We do a mixing test where we mix normal pooled plasma with all factors present with the patient’s plasma. If the issue is deficiency, then NPP should solve the problem, and clotting time will correct and return to normal. If it’s an inhibitor, even the newly added factor will get inhibited, and APTT will still remained prolonged

How does heparin related to APTT?

Heparin is a thrombin inhibitor which can also prolong APTT

What is a D-dimer?

A product of fibrin breakdown, telling us that fibrin was made and then broken down

What does a D-dimer represent and how can we use D-dimers?

Represents the formation then breaking down of fibrin(aka a coagulation cascade starting, completing, and ending), and we can use it to rule OUT DVT and DIC. If D-dimers are low, it’s likely that the patient doesn’t have DVT or DIC

What is DVT and what is DIC?

DVT: Deep Vein Thrombosis, or a big fibrin mass formed in a deep vein that blocks blood flow and can detach and cause PE

DIC: Disseminated Intravascular Coagulation, or a condition where lots of clotting happens throughout the body, using up platelets and clotting factors, causing paradoxical bleeding all over the body

How does primary hemostasis work?

1. Blood vessel injury exposes subendothelial collagen

2. Circulating vWF and on platelets help act as bridge between collagen and platelets

3. These platelets use more factors to recruit even more platelets and form a plug

In concept, what does it mean to add an “a” after a factor? Eg. Factor XII and XIIa?

Adding an “a” means the factor has been activated

What form are factors in as they circulate normally around in blood?

Zymogen(inactive) form

Why is it called “intrinsic pathway”

Because blood(within the blood vessel) comes into contact with the damaged surface it’s not supposed to normally touch

How does the intrinsic pathway start, and what are the 3 players involved in the first step of the intrinsic pathway, and what are they?

1. Factor XII: zymogen

2. Prekallikrein: zymogen

3. High molecular weight kininogen(HMWK): cofactor to help factor XII and Prekallkrein interact

What happens when factor XII and prekallikrein interact?

It turns factor XII into factor XIIa, and XIIa helps activate prekallikrein into kallkrein which will also help turn more factor XII into factor XIIa

Finish the intrinsic pathway: happens after XIIa is formed?

1. XIIa activates XI into XIa, which activates IX into IXa

2. Factor IXa can’t work on it’s own, it needs factor VIIIa, Ca2+, and a phospholipid surface(from platelets) to form a complex to activate factor X into factor Xa

How does the extrinsic pathway start, and why is it called the extrinsic pathway?

It’s called that because the cells surrounding the blood vessel get exposed to blood. These cells contain factor III, also called tissue factor, which binds factor VII in the blood, turning it into factor VIIa

What does factor VIIa do?

It forms a complex with tissue factor which needs Ca2+ and then also activates factor X into factor Xa

The common pathway starts with factor X becoming Xa, what happens after that?

• Factor Xa needs a partner, Factor Va, Ca2+, and a phospholipid surface to form the prothrombinase complex

• Prothrombin(aka factor II) gets cut by the complex and becomes factor IIa, also called thrombin

Once we now have thrombin, what does it do?

1. Thrombin: Cuts factor 1(fibrinogen) into fibrin, the mesh that forms the clot

2. Activates factor XIII into XIIIa. Factor XIIIa is what crosslinks fibrin

3. Will amplify the pathway: V into Va, VIII into VIIIa, basically positive feedback

Alternate names of

Factor I, II, III

I: Fibrinogen

II: Prothrombin

III: Tissue factor

What is the cell-based model of coagulation and what does it tell us?

• It’s a model that tells us the extrinsic pathway is much more important than the intrinsic pathway. It says that secondary hemostasis occurs via 3 phases: Initiation where a TF-bearing cell meets factor VII(so like the normal extrinsic pathway), and this ends up forming Xa which binds with some small existing Va, which makes a small burst of thrombin. This thrombin then goes to activated platelets nearby and starts the amplification phase where the thrombin boosts Va, VIIIa, and XIa(which activates more IXa). This ends in the propogation phase where IXa, VIIIa can now form a big burst of Xa which binds the now increased Va and leads to prothrombinase being formed and creating even more thrombin and begins making the fibrin clot.

Explain the differences in clinical signs of a patient with primary and secondary hemostatic defect

Primary hemostatic defect: Petechiae, small superficial ecchymosis and mucosal bleeding.

• This is because petechiae is due to the small, minor capillary damages we get day to day that platelets typically heal no problem, we get superficial ecchymoses from minor bruises because platelets normally patch these but regardless they don’t become massive because the coagulation cascade deals with it. Mucosal bleeding is common because in places like gums and nose lining and GI, we have lots of capillaries

Secondary hemostatic defect: Delayed bleeding, large, deep ecchymosis/hematoma and hemathrosis

• This is because platelets can initially plug a wound, but it’s not enough, without the fibrin mesh forming, bleeding will appear again. Deep ecchymoses and hemarthrosis(bleeding into joints) are injuries that require stronger fibrin meshes and platelet plugs can’t hold

If CBC is all normal except thrombocytopenia with normal PT and aPTT, and the condition is acquired, what is it likely to be?

Immune thrombocytopenia

In terms of platelets, how can increased portal blood pressure impact the spleen?

• Blood backs up into the splenic vein and physically enlarges the spleen allowing it to sequester more platelets

What is an A/G ratio, and what does it mean if it’s reversed?

• Ratio between albumin and globulin, which should be above 1, but reversal indicates that either albumin production has decreased, globulin production has increased, or both