Lecture 19 Coagulation Interferences & Validating Lab Results

Question 1

What is the most important pre-analytical steps to validating coagulation lab results?

Answer 1

Proper patient identification is crucial.

See slide 3 for more details.

Question 2

Why should the tourniquet be removed within 1 minute (collection techniques)?

Answer 2

Tourniquet should be removed within 1 minute of its application to avoid blood stasis and accumulation of Factor V, VII,VIII, XII and VWF which may falsely shorten clot based coagulation results.

Question 3

What do you do if the phlebotomy has been unsuccessful?

Answer 3

If phlebotomy unsuccessful tourniquet should be released for 2 mins then reapplied for repeat attempt.

Question 4

What issue can repeated probing with the needle cause with coagulation testing results?

Answer 4

1. Tissue fluid from repeated probing may affect coagulation results as Tissue Factor responsible for activating extrinsic and intrinsic pathways.
2. Samples from traumatic collection may be falsely shortened and unreliable

Question 5

What kind of tubes should coagulation samples be collected in?

Answer 5

Coagulation samples should be collected in plastic blue-stopper sterile evacuated tubes with 3.2% buffered sodium citrate anticoagulant.

Question 6

What kind of tubes are not acceptable for coagulation samples? How can they be modified to be suitable?

Answer 6

1. Uncoated glass tubes are not suitable as their negative charge activates platelets and plasma procoagulants.
2. Coated siliconized glass tubes are available and suitable but not preferable due to breakage and exposure to blood borne pathogens.

Question 7

What if you need to draw blood above the intravenous line?

Answer 7

Drawing above Intravenous line
I.V. must be shut off for a period of time (usually approx 2 mins-check with facility policy) before drawing above I.V.

Question 8

What do you need to do if you are drawing blood from arterial and venous lines?

Answer 8

Drawing blood from arterial and venous lines:
Must ensure proper discard is removed (usually approx 5ml.- check with facility policy) before drawing blood for lab analysis as these lines usually contain heparin to prevent clotting and will elevate coagulation results.

Question 9

What kind of bad phlebotomy practices causes hemolysis?

Answer 9

1. Needle bore too small
2. Quickly pulling back on plunger of syringe
3. Forcing blood from syringe into tube
4. Not removing excess alcohol or H2O from venipuncture site.
5. Vigorous shaking of tube. Never shake !!

Hemolysis affects coagulation.

Question 10

Does hemolysis increase or decrease coagulation results?

Answer 10

No firm data. Can depend on instrument method of measurement and manufacturer.
Textbook says hemolysis may cause falsely shortened results due to activation of coagulation factors and platelets that generated FVIIa and thrombin.

Question 11

What type of measurement is less sensitive to hemolysis?

Answer 11

In general mechanical measurement is less sensitive to optical interference caused by hemolysis when compared to nephlometry or turbidometric measurement.

Question 12

How can icteric or lipemic samples affect coagulation results?

Answer 12

1. Icteric, lipemic samples can affect times in instruments using photometric methods. Some labs may ultracentrifuge samples to separate lipids from plasma.
2. Similar to hemolysis no firm data to indicate whether lipemic, icteric samples will increase or decrease coagulation results.
3. Your textbook page 800 indicates lipemia may falsely prolong results on OD instruments due to interference with light transmittance.
4. Your textbook page 800 indicates Icteric samples produce prolonged clotting times due to inadequate liver factor production; may also interfere with OD instruments

Question 13

What measurement method is the least sensitive to lipemic/icteric interferences?

Answer 13

Similar to hemolysis mechanical measurement of lipemic/icteric samples is less sensitive to interferences than compared to optical measurement.

Question 14

How will partially clotted specimens affect coagulation results?

Answer 14

Partially clotted specimens may cause falsely shortened coagulation results due to activation of coagulation Factors VIIa and thrombin.

Question 15

How will fully clotted specimens affect coagulation results if tested and not noticed?

Answer 15

Fully clotted specimens similar to that seen in serum tubes will give falsely elevated results due to consumption and depletion of coagulation factors. Fully clotted specimens contain no fibrinogen.

Question 16

Is proper centrifugation important to remove platelets? What are the time/rpm spun at?

Answer 16

Proper centrifugation important to remove platelets- (ie 15 mins. at 3000 rpm).
Some Shared Services sites using 4 mins. at 5000 rpm. Must be validated at each site.

Question 17

What centrifugation quality measures are taken to ensure proper coagulation testing results?

Answer 17

1. Centrifuges should be periodically checked for proper time, speed and function.
2. Weekly platelet count on plasma part of quality control. Should be less <10 x 109/L to reduce platelet interferences.

Question 18

Is order of draw important? When should the coagulation testing tube be drawn?

Answer 18

1. Vacutainer system- Light blue Na Citrate samples for coagulation testing should be drawn according to site SOP.
2. Usually first tube or immediately after a non-coagulated tube.

Question 19

What tubes if drawn before the coagulation testing tube would contaminate needles and falsely elevate results?

Answer 19

Coagulation samples collected after Heparin or EDTA tubes may contaminate needles and can cause falsely elevated results.

Question 20

What is the tube of choice for coagulation testing and why?

Answer 20

Trisodium citrate is anticoagulant of choice because preserves best labile factors V and VIII.

Question 21

What type of tube is best for samples with higher hematocrit and most suitable for platelet aggregation studies and monitoring heparin therapy?

Answer 21

Sodium citrate available in 3.2% and 3.8%. 3.2 % is preferred as will reduce interference in samples with higher hematocrit
Most suitable anticoagulant for platelet aggregation studies.
Citrate specimen more sensitive to effects of heparin therefore preferred to monitor heparin therapy.

Question 22

Why is an EDTA tube not suitable for coagulation testing?

Answer 22

EDTA not suitable anticoagulant because inhibits thrombin-fibrinogen reaction.
Factor V and FVIII not stable in EDTA.

Question 23

Why is an heparin tube not suitable for coagulation testing?

Answer 23

Heparin tube not suitable because it is a strong anti-thrombin. Used as a anticoagulant.

Question 24

What is the standard ratio and amounts of blood vs anticoagulant?

Answer 24

Proper filling and ratio of blood is critical.
Standard ratio is 0.5ml anticoagulant plus 4.5ml blood. Ratio is 1:9.

Question 25

What is the effect on coagulation results if the tube is not fully filled?

Answer 25

Improper filling (tube not filled) lead to excess anticoagulant which interferes with recalcification phase of testing and falsely high results. 90% fill is required.

Question 26

If Hct values are high or low what is the impact on coagulation testing results?

Answer 26

If RBC’s high may need to adjust Na citrate volume If Hct >0.550 result will be falsely high If Hct <0.200 no studies to suggest results will be affected

Question 27

Why do samples with a high Hct need anticoagulant removed before testing?

Answer 27

1. Samples with high Hct >55% contain low plasma volume therefore high concentration of Na Citrate 2. High Na Citrate concentration interfere with recalcification stage of testing causing falsely elevated results. 3. Sample needs to be recollected in Na Citrate tube containing a decreased amount of anticoagulant

Question 28

What quality analytical variables need to be considered to get reliable coagulation testing results?

Answer 28

1. Proper instrument calibration. 2. Proper quality control. Most labs run 3 controls (normal and two elevated result) on each shift. 3. All accredited labs participate in external QC program.

Question 29

How should samples for coagulation testing be stored?

Answer 29

1. Samples should be stored in an upright position with stopper intact prior to testing. Uncapping sample will alter pH and factor viability. 2. Samples should be stored at room temp (between 18 and 24ºC) as refrigeration will activate FVII, destroy platelet activity by activation and cause precipitation of large VWF multimers.

Question 30

What happens if samples were stored above 24degC for coagulation testing?

Answer 30

Samples should never be stored at temperatures >24ºC as heat causes deterioration of Factors V and VIII

Question 31

What is the storage requirements specifically for PT/INR testing?

Answer 31

Samples for PT/INR may be held at 18-24ºC uncentrifuged or centrifuged and tested within 24 hrs. from time of collection provided tubes remain closed

Question 32

Why must APTT testing occur within 4 hrs after being held at 18-24C?

Answer 32

1. Samples for APTT may be held at 18-24ºC uncentrifuged or centrifuged but must be tested within 4hrs. from time of collection provided they do not contain unfractioned heparin (UFH) 2. APTT testing within 4 hrs. is performed to reduce deterioration of labile Factors V and VIII

Question 33

If samples containing unfractioned heparin (UFH) are needed for APTT testing what must be done and why?

Answer 33

Samples containing UFH must be centrifuged within 1hr. from time of collection and tested within 4hrs. Plasma may remain on packed cells. Centrifugation within 1hr. is performed to reduce the effect of heparin neutralization by platelet granule Platelet Factor 4 (PF4).

Question 34

If you can't perform coagulation testing within the required times what must be done to test in future?

Answer 34

1. Samples that cannot be performed within the previously described intervals must be spun immediately. 2. The supernatant platelet poor plasma (PPP) must be transferred by plastic pipette to plastic freezer tube and sealed. **Non-siliconized glass must never be used as contact with glass will activate coagulation cascade.

Question 35

How long can samples containing platelet poor plasma (PPP) be frozen for and at what temperatures?

Answer 35

Samples containing PPP) may be frozen at minus 20ºC for up to 2 weeks and at minus 70ºC for up to 6 mos.

Question 36

How do you thaw frozen PPP samples for testing?

Answer 36

All frozen coagulation samples should be rapidly thawed at 37ºC to prevent deterioration of Factors V and VIII, mixed well and tested within 1 hour.

Question 37

What analytical checks should one do to eliminate or reduce erroneous errors?

Answer 37

1. Re-checking sample for proper filling and signs of interference (ie hemolysis, lipemia, icteric etc) 2. Checking sample for clot after repeat testing 3. Phoning for clinical, medication history. Heparin, factor deficiency, liver disease, lupus inhibitor, antibiotics can all affect results. 4. Follow-up of erroneous results will also include repeat testing of sample and possible re-collect to confirm results. 5. If results elevated and no explanation, a 1:1 mixing study should be performed to separate factor deficiency from lupus or factor inhibitor. 6. Delta checks should be performed to detect labelling errors and change in patient’s condition. 7. Proper recording of results: paper or electronic.