What does phospholipid do in the PT and PTT?

phospholipid

Q. I have a quick question about coag lab tests. In the tests that you are adding phospholipid (like the PTT), what exactly is the phospholipid doing?

A. It’s just providing a surface for the coagulation factors to sit on! Many of the coagulation factors need a phospholipid surface to sit on in order to work.

Normally, the platelets provide that surface (they have phospholipids in their membranes) – but you’ve taken the platelets out of the test tube before you do the coagulation lab tests – so you need to add them back in if you want the whole cascade to run.

You also add phospholipid in the PT (INR)! It’s part of the thromboplastin molecule. Thromboplastin is just a tissue-factor-like substance plus phospholipid, all wrapped up in one reagent.

A few of the coag tests don’t require a phospholipid surface. The TT, for example, doesn’t need phospholipid; you’re just adding thrombin and seeing how fast it can convert fibrinogen to fibrin – and that single reaction doesn’t need phospholipid to work. Also, the fibrinogen assay doesn’t require phospholipid because it just measures the amount of fibrinogen.

Why do the INR and PTT measure different pathways?

test tubeCoagulation questions seem to come up all the time! Here’s a good one from one of our readers.

Q. In both the PT and PTT we add thromboplastin, right? So how come the PT measures the extrinsic pathway and the PTT measures the intrinsic pathway?

A. This is a great question because it really gets at the underlying concepts of the PT (INR) and PTT. When I was a medical student, I never really thought about why the INR only measured the extrinsic pathway and the PTT measured only the intrinsic pathway. I just memorized the substance added to the test tube in each test, and the pathway the test measured. Later on, though, I realized I didn’t have a clue as to why the tests measured the pathways they did.

Before we get into the reasoning behind the tests, a quick correction is in order. We don’t add thromboplastin in both the INR and PTT. In the INR, you add thromboplastin, and in the PTT you add phospholipids (not thromboplastin). It turns out thromboplastin is a substance that contains both phospholipids AND a tissue-factor-like substance. That’s why they call the assay the “partial thromboplastin time” – because you only need to add part of the thromboplastin reagent (the phospholipid part) to get this test to run.

To understand why the PT measures just the extrinsic pathway and the PTT measures just the intrinsic pathway, you need to know what activates these pathways in the body. The extrinsic pathway is activated by tissue factor. The intrinsic pathway can be activated by a bunch of things, the most important of which is thrombin.

Why the INR measures the extrinsic pathway

To get blood in a test tube to form fibrin along the extrinsic pathway, you need to add some tissue-factor-like substance. Also, since you removed the platelets and calcium before running the test, you need to add those things back into the test tube (the coagulation system needs a phospholipid surface, normally provided by platelets, and calcium to run). Thromboplastin is a substance that contains both phospholipids and a tissue-factor-like substance. Add thromboplastin and some calcium, and the blood in the test tube will form fibrin via the extrinsic pathway.

Why the PTT measures intrinsic pathway

To get blood in a test tube to form fibrin along the intrinsic pathway, you don’t need to add any tissue-factor-like substance (if you do, the extrinsic pathway will be activated!). All you need to do is add back what you took out of the blood (phospholipids and calcium), as well as something like silica or kaolin to activate the intrinsic pathway (normally, thrombin does this job in vivo), and you’ll form fibrin along the intrinsic pathway. This is actually why the intrinsic pathway was named the way it was: everything you need to get the pathway to run is “intrinsic” to the blood. The extrinsic pathway requires something “extrinsic” to the blood (tissue factor) for it to run.

Bottom line

The INR activates the extrinsic pathway because in this test you add thromboplastin (which contains both a tissue-factor-like substance and phospholipids) to the test tube. The PTT activates the intrinsic pathway because in this test you add just phospholipids to the test tube – and without tissue factor around, fibrin is formed along the intrinsic pathway.

Hematopathology Quiz Part 3

pencil
Here is the final installment of a really nice quiz one of our readers sent (check out the first and second installments).

This is a quiz recently given to medicine, pediatrics and pathology residents rotating through hematopathology. Give it a try and see how many you get right! Make sure you check out the answers and nice explanations (if I do say so myself) at the end.

1. A 24-year-old female presents with prolonged PTT which did not correct following incubation for 2 hours with control plasma. Which of the following is the most likely diagnosis?

A. Hemophilia A
B. Antiphospholipid antibody syndrome
C. Factor V Leiden
D. Von Willebrand disease

2. A “dry tap” on bone marrow aspiration can be seen in all of the following disorders EXCEPT:

A. Chronic myeloid leukemia
B. Hairy cell leukemia
C. Idiopathic thrombocytopenic purpura
D. Polycythemia vera

3. All-trans-retinoic acid is the treatment of choice for which of the following leukemias?

A. Acute promyelocytic leukemia
B. Acute lymphoblastic leukemia
C. Chronic lymphocytic leukemia
D. Chronic myeloid leukemia

4. Which of the following stains is used for supravital staining of polychromatophilic red cells?

A. New methylene blue
B. Oil red O
C. Leishman stain
D. Masson’s trichrome

5. Giant platelets are seen in:

A. Glanzmann thrombasthenia
B. Bernard-Soulier syndrome
C. von Willebrand disease
D. Thrombotic thrombocytopenic purpura

6. The leukemic cells in large granular lymphocyte leukemia are positive for:

A. CD33
B. CD 117
C. CD56
D. CD34

7. A 65-year-old female presents with fatigue. A blood smear shows abnormal lymphocytes, which are positive for CD5, CD19, and CD20, but negative for CD23. Cytogenetic analysis reveals a t(11;14). What is the diagnosis?

A. Hairy cell leukemia
B. Mantle cell lymphoma
C. Marginal zone lymphoma
D. Chronic lymphocytic leukemia

8. Serum tryptase is elevated in:

A. Hairy cell leukemia
B. Mast cell leukemia
C. T-cell acute lymphoblastic leukemia
D. Biphenotypic leukemia

 

 

 

 

Scroll down for the answers…

 

 

 

 

 

 

 

1. B. If you have a prolonged PTT, and you’re wondering what the cause is, one thing you can do is a mixing study, which is what this question is getting at. In a mixing study, you add a little normal plasma to the PTT (along with the patient sample) and then run the PTT again. If it “corrects” (meaning that the PTT is now normal), then it means the patient was missing something that the normal plasma provided (usually the thing that’s missing is factor VIII or IX). If it doesn’t correct (meaning that the PTT is still prolonged), then there is something in the patient sample that is inhibiting the test (usually the thing that’s inhibiting the test is an antiphospholipid antibody). So for the answers to the question: the correct answer is B (since the PTT didn’t correct). In hemophilia A, a mixing study would make the PTT correct, so A is wrong. Patients with factor V Leiden or von Willebrand disease don’t have a prolonged PTT, so C and D are wrong.

2. C. A “dry tap” means that you get the needle in the marrow cavity, and when you pull back on the syringe, no marrow is drawn out. This usually means that there is fibrosis in the marrow, which is something you can see in pretty much any myeloproliferative disorder, particularly in the end stages (so A and D could produce a dry tap). In hairy cell leukemia (B), there are little threads of fibrosis surrounding each hairy cell. It gives the marrow a “chicken-wire” appearance. Patients with ITP (C) don’t have fibrosis in the marrow. The problem in ITP is that the body is attacking its platelets – it has nothing to do with fibrosis in the marrow – so C is correct.

3. A. All-trans-retinoic acid (ATRA) is a treatment that was developed for acute promyelocytic leukemia (APL). In APL, the retinoic acid receptor is screwed up, and the malignant cells get “stuck” in the promyelocyte stage. This is not good, because if you give a patient with APL regular chemotherapy, which tends to bust open cells, you’ll release all the promyelocyte granules (which contain pro-coagulant substances), putting the patient at risk for disseminated intravasular coagulation. ATRA overcomes this maturation block, allowing the malignant promyelocytes to mature into myelocytes, then metamyelocytes, then neutrophils. Then you can give the patient regular chemotherapy without the risk of DIC. ATRA doesn’t work in any of the other leukemias listed, because those leukemias don’t have a problem with the retinoic acid receptor.

4. A. Supravital staining means that you do the stain on liquid (not fixed and dried) blood. Supravital stains are used only in certain settings, like when you’re looking for Heinz bodies, or when you’re counting reticulocytes (polychromatophilic cells). The supravital stain used to stain polychromatophilic cells is new methylene blue (A).

5. B. Both Glanzmann thrombasthenia and Bernard-Soulier syndrome are hereditary platelet disorders in which platelet receptors are deficient or defective, and patients bleed excessively. In Glanzmann, IIb-IIIa is deficient, meaning that the platelets can’t bind fibrinogen very well, which means they can’t aggregate well. In Bernard-Soulier, Ib is abnormal (meaning that platelets can’t bind von Willebrand factor very well, and can’t adhese to the subendothelium very well). It turns out that patients with Bernard-Soulier (but not Glanzmann) have big platelets, too. So B is correct. Big platelets are not seen in von Willebrand disease (C) or TTP (D).

6. C. The lymphocytes in large granular lymphocyte leukemia are T cells. They are typically positive for CD8, CD 16, and CD56 or CD57. CD33 (A) is a marker seen mostly on myeloid cells. CD117 (B) is present on hematopoietic stem and progenitor cells, and CD34 (D) is present on hematopoietic stem cells, endothelial cells, mast cells and some dendritic cells.

7. B. The t(11;14) is characteristic of mantle cell lymphoma, and not of any of the other types of lymphoma listed. Also, mantle cells tend to be CD5, CD19 and CD20 positive, but CD23 negative. The cells of hairy cell leukemia (A), marginal zone lymphoma (C) and CLL (D) have different immunophenotypes.

8. B. Mast cells contain tryptase (which is a serine protease) in their little granules. Hairy cells (A), T-cell ALL (C) cells, and the cells of biphenotypic leukemia (D) are all negative for tryptase.