Module 12a. Hematology Laboratory. Clot Retraction Test PDF

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MODULE 1: PLATELET COUNT AND PLATELET FUNCTION TESTSTopic Outline:I. CLOT RETRACTION a) Standard Procedure b) Interpretation of results c) Other Methods for Clot Retraction d) Conditions that affect clot retractionLearning Outcomes: Discuss the principle of Clot retraction Test Perform Clot Retracti...

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MLS12Aa. HEMATOLOGY (LAB) MODULE 1: PLATELET COUNT AND PLATELET FUNCTION TESTS

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Topic Outline: I. CLOT RETRACTION a) Standard Procedure b) Interpretation of results c) Other Methods for Clot Retraction d) Conditions that affect clot retraction Learning Outcomes: 1) Discuss the principle of Clot retraction Test 2) Perform Clot Retraction 3) Correlate results with related conditions LEGENDS:  Module Packet  Instructor’s Discussion

Ultrastructure of Platelet 1. Peripheral Zone: a. Plasma Membrane b. Glycocalyx Is where Glycoproteins are seen Outermost portion c. Submembranous Membrane or OCMS 2. Sol-gel Zone: support of the cell structure a. Microtubules Tubulin – maintains the platelet discoid shape b. Microfilaments Actin Myosin When actin and myosin interact with each other, they form Thrombosthenin or Actomyosin 3. Organelle Zone: a. Alpha granules b. Dense granules Contains non-protein mediators 4. DTS: sequestration of internal calcium for the control of cell activation

Note:  What phosphatidyl is can be seen in platelets that “flip ups” during the platelet activation?  Phosphatidylserine  Aside from the alpha and dense granules, what other cell organelles are found in the platelets?  Mitochondria, Lysosomes, Perixosomes  What is the purpose of Thrombosthenin?  It is the protein responsible for the clot retraction

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MLS12Aa. HEMATOLOGY (LAB) MODULE 1: PLATELET COUNT AND PLATELET FUNCTION TESTS Within 24 hours

PRINCIPLE: Determines the length of time required for firm clot formation by both platelets and fibrinogen.

 Clot retraction measures the ability of the blood to retract, which means that it decreases clot size during formation of a hemostatic plug.  When blood is allowed to be placed in a glass tube without an anticoagulant at 37oC the clot will begin to shrink and "retract" from the walls of the tube thus expressing the serum.  measures the percent of serum yielded in the tube.  Thrombosthenin, released by the platelets, is responsible for clot retraction as well as other contractile proteins- actin and myosin, because of this we can use this test to determine both platelet quantity as well as it's function.  Clot retraction is directly proportional to the platelet and inversely proportional to the fibrinogen concentration.  Normally clot retraction begins within 30 seconds after the blood has clotted.  Measures platelet quantity and quality After 30 seconds: At the end on 1 hour At the end of 4 hours

Start of clot retraction or will start to shrink There should be appreciable clot retraction Almost complete retraction  More serum is seen

with platelets or fibrinogen if clot is not completed

 A high red cell count limits the degree of retraction because of the large volume of RBCs within clot.  4. An increased RBC fall-out at the bottom of the tube, indicates slightly decreased in fibrinogen and should be reported and noted on the result.

CLOT RETRACTION (CLOT RETRACTION TIME)

 When a blood sample is not anticoagulated (meaning we’re trying to take out your serum), the blood in the serum started to clot thus forming a giant clot in vivo. What does this have to do? After a while, normally your platelets and fibrinogen function normally your clot will start to decrease in size if it’s the blood in tube without anticoagulant tube at 37C leaving your serum

Clot retraction must be completed  Otherwise, there would be some deficiencies

Decrease in clot size:   

Blood in tube without an anticoagulant at 37oC Expresses serum GOAL: Measure the yield serum

CLOT RETRACTION IS INFLUENCED BY: The number and activity of platelets

Concentration of fibrinogen

Packed Cell Volume

Platelets are the forcegenerating components of clotretraction.

Fibrinogen is needed for fibrin formation that will later on trap platelets and act together with them to retract the clot and restore the damaged blood vessel walls.

High red cell counts limit the degree of retraction because of the large volume of red cells within the clot.

Once the hemostatic plug has been formed the actin and myosin portion of the platelets act together with fibrin to shrink the size of the plug thus causing

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MLS12Aa. HEMATOLOGY (LAB) MODULE 1: PLATELET COUNT AND PLATELET FUNCTION TESTS retraction that will aid in vascular wall and tissue repair. Actin & Myosin + Fibrin = shrinks the size of the plug

CRT is directly proportional to clot retraction  If the number of platelets is small, they could not shrink at the right amount of time  If less platelets, it will take a long time for the clot to retract  If more platelets, shortens the amount of time to retract

Consumed to produce more fibrin  If less fibrinogen = less fibrin activated = prolonged time to retract

Limits the degree of retraction due to presence of red cells in clot  In polycythemia vera,

When they shrink, the tissue also shrinks so that the hoel or damaged portion will start to close out or heal.  Purpose of clot retraction: To close out the tissue or damaged the blood vessel by shrinking the size of platelet plug  If platelet is adequate in number, but not functioning properly = prolonged time to retract CRT Relies heavily on the number and function of the platelet

clot retraction takes long because of the presence of several red cells  PCV plays a major role in clot retraction  Presence of red cell = platelets take a long time to retract  Red cells are used to form clots

CRT is inversely proportional to fibrinogen concentration  Fibrinogen is needed to make more fibrin so if there is less fibrinogen, more fibrin = clot retraction is achieved  More fibrinogen , less fibrin is converted = clot retraction will be dysfunctional

 When your platelets and fibrin start to form platelet plug, platelet retraction will occur.  What happens during platelet contraction? In the 3rd pic, each platelet adheres to each strand of fibrin. Later on, actin and myosin will take the fibrin and starts to stiffen and contract. Once it starts to contract, it will pull the fibrin and platelet plug, thus starting clot retraction. While pulling the platelet plug, they’re pulling the vessel wall to start tissue repair

 Why there is a need to shrink the size of the plug? The platelet plug is only covering the wounded area and there is not enough to just cover the leak of the wounded area so you need to take the ends of the blood vessel of tissue so you could try to close it out.  How does the actin & myosin close this out? By retracting or shrink in size so that they could pull the ends of the tissue or damage vessel.

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MLS12Aa. HEMATOLOGY (LAB) MODULE 1: PLATELET COUNT AND PLATELET FUNCTION TESTS  If your platelets or fibrinogen are not functioning normally, you will see a loose clot and some RBC will start to fall off the tube because it has not clotted properly

STANDARD PROCEDURE FOR CLOT RETRACTION:

PROCEDURE

1

Withdraw 3 ml of venous blood.

2

Place 1 ml of blood into each of 3 glass test tubes, centrifuge tube, and immediately incubate in a 37oC water bath. Set a timer for 1 hour.

3

At 1 hour, observe the clot and record results.

4

Inspect the tubes at 2, 4, and 24 hours, observe and record results.

5

Examine the tubes for retraction after incubation.

6

Separation of the clot from the test tube is complete retraction (+4).

7

If clot retraction occurred within 2-4 hours: NORMAL; between 4-24 hours: POOR.

 Note: In performing Clot Retraction, remember that this is very easy and simple. First of all, gain a 3 ml of blood using the venipuncture procedure. This 3ml of blood is then delivered to the 3 test tubes, 1 ml each. Then test tubes are placed in the water bath at 37°C for 1 hour. After 1 hour, take it out from the water bath and set aside at room temperature. Check for clot retraction at 1 hour, 2 hours, 4 hours and 24 hours. After 24 hours, the serum yielded is measured to correlate with the clot retraction time.

Determination of Result: Approximate amount of shrinkage in the clot. Either using percentage or using the grading system.

SOURCESOFERRORWHENPERF ORMI NGCL OT RET RACT I ON  

Shaking or jarring of the tube of blood should be avoided. This may lead to a shortened clot retraction time. Certain anemic patients with a low hematocrit value show increased clot retraction due to the formation of a small clot.

 This is what your blood sample would look like if not placed in anticoagulated tube. Later on, the blood will form blood clot and shrink in size leaving the serum in the tube

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MLS12Aa. HEMATOLOGY (LAB) MODULE 1: PLATELET COUNT AND PLATELET FUNCTION TESTS

I NTERPRET AT I ONOFRESUL T S: The clot will retract from the walls of the tube until the red cell mass occupies 50% of the total volume of blood in the tube. There is a variable degree of retraction or there is no retraction at all. Grades as follows: no serum extruded  Red cell is still intact no clotting occurred 5 – 10% serum extruded

0 1+ 2+

10 – 20% serum extruded

3+

20 – 35% serum extruded

4+

35 – 50% serum extruded

RESULTS ARE REPORTED AS THE LENGTH OF TIME IT TOOK FOR THE CLOT TO RETRACT 2 – 4 hours

normal

After 4 hours

Poor

After 24 hours

No retraction

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 This shows what tube would look like after an hour in an actual setting  The graph tells you all about these tubes.  Vertical line (y-axis) shows the percent of the original volume  Horizontal line (x-axis) shows the time in minutes  This top (WT) or normally retracted blood would show that the volume would approximately be lowered or would retain its volume as time goes on  The bottom (-/-), when the clot will not retract you could see that the volume of the blood sample is not the same as the one where the clot has retracted

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MLS12Aa. HEMATOLOGY (LAB) MODULE 1: PLATELET COUNT AND PLATELET FUNCTION TESTS

OTHER METHODS FOR CLOT RETRACTION HI RSCHBOECKMET HOD

STEF ANI NI –DAMESHEKMETHOD

MACF ARLANEMET HOD

“ Si n gl eT e s tT ub eme t h od ” Makes use of castor oil

Makes use of a single test tube

PROCEDURE 1

Add one drop of Castor oil

1

Withdraw 3ml of blood

1

Place 5ml of fresh blood in a calibrated tube

2

Add one drop of blood

2

Transfer in a Wasserman Tube

2

Place a glass rod into the tube

3

Incubate at 37oC or place at RT and allow 2 hours to elapse

3

 Purpose of glass rod: to remove the clot since during the set up dira ga bind ang clot. (F XII)

4

After 2 hours observe the clot

4

Incubate at 37oC in a water bath

5

Record observation as: No retraction Partial Retraction Complete Retraction

5

Examine every 5-10 minutes for retraction

6

Observe the appearance of the clot. Record as firm or soft

6

After coagulation, leave the tube at room temperature for retraction

7

After retraction has taken place, remove the glass rod to remove the clot

8

measure the amount of serum left on the tube directly against the calibration of the tube

Fit a cork at one end of the glass rod

3

Start the timer & observe dimpling

Normal Values: 15 – 45 mins. < 15 mins: Thrombotic Tendency >45 mins: Hemorrhagic Tendency Incomplete or Delayed occur in Thrombocytopenia & Glanzmann Thrombasthenia

END PRODUCT: CLOT

NOTE: Qualitative Method: Hirschboeck Quantitative Method: Stefanini, Macfarlane

Computation: %CR= ml of serum/ml of blood x 100

Normal Value: 44-67%

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MLS12Aa. HEMATOLOGY (LAB) MODULE 1: PLATELET COUNT AND PLATELET FUNCTION TESTS CONDITIONS THAT AFFECT CLOT REACTION TIME   

Fibrinogen deficiency (congenital or acquired) = prolonged CRT Thrombocytopenia (less than 100,000) = prolonged CRT Thrombosthenia  Platelet would not function properly. Platelets could not pull or firm the fibrin therefore leading to prolonged or no CRT



Polycythaemia vera = prolonged CRT  Red blood cells are needed to form a clot. If too much RBCs in the clot, platelets will have a hard time to shrinking the clot leading to prolonged CRT  Unlike in anemia, where clot retraction is increased / shorten because you have red cells present in the body  to remove the clot since during the set up dira ga bind ang clot. (F XII)  Prolonged, because RBC count limits the degree of clot retraction



Reduced clot formation: Glanzmann thrombasthenia, DIC, hypofibrinogenemia, dysfibrinogenemia (small clot with increased red blood cell “fall-out”) = prolonged CRT o DIC = prolonged o Hypofibrinogenemia = prolonged o Dysfibrinogenemia = prolonged

Re v i e wa ndSu mma r y : 1) 2) 3) 4) 5) 6)

What is the normal value for Hirschboeck method? 15-45 mins Normal value for MacFarlane method? 44-67% Blood retraction relationship with platelets? Directly proportional Blood retraction relationship with hematocrit? Inversely proportional Product observed in Hirschboeck method? RBC dimpling How many hours do we incubate blood for standard blood retraction? 1 hour 7) How many hours do we incubate for Stefanini-Dameshek method? 2 hours 8) What method uses Wasserman tube? Stefanini-Dameshek method 9) Other name of Stefanini-Dameshek method? Single Test Tube Method 10) Normal clot retraction begins in how many seconds? 30 seconds 11) If we shake the test tube with blood what will happen to the CRT? Shortened CRT 12) Other 2 Methods for Blood Retraction: Tocantin’s Method and Budtz Olsen Method 13) When a normal blood clot contracts it expresses the serum 14) It Is directly parallel to platelet count. Retraction 15) TRUE / FALSE. If retraction has taken place the clot will shrink and attach to the glass rod. TRUE

Note: Tocantin’s Method in platelet count (Direct Method) is different from Tocantin’s method in blood retraction

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MLS12Aa. HEMATOLOGY (LAB) MODULE 1: PLATELET COUNT AND PLATELET FUNCTION TESTS

~END OF MODULE 3~

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