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The interpretations below are provided by Donna Castellone, MS, MT (ASCP) SH for Aniara Diagnostica, Inc.

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A patient referred to Coagulation Laboratory after receiving a diagnosis of possible von Willebrand disease due to low Factor VIII activity, low vWF activity and a normal antigen. New samples were drawn at the laboratory, processed and retested. All results were within the normal range. What can cause this discrepancy? Was the wrong patient tested? An analytical error – problem with the reagent? Post-analytical- wrong patient result?

Most errors occur in the preanalytical phase. A big problem with coagulation testing is the instability of the factors. We are working with enzymes and unstable proteins, so having a sample that is tested within the recommended stability time is important to ensure accurate results.

Coagulation factor assays are very important for diagnosing, treating, and monitoring inherited and acquired bleeding disorders. Therefore, it is necessary to evaluate the effects of pre-analytical variables for sample storage. The important pre-analytical variables primarily comprise sample collection, transportation, storage time, and temperature. Unsuitable samples can lead to unreliable factor activities and interfere with clinical decisions.¹

GUIDELINES:

CLSI document H-21, 6th edition states that whole blood samples should be transported at room temperature. Transporting samples on ice can cause cold activation of FVII, loss of vWF, FVIII and platelet disruption. Samples sent for PT testing may be stable refrigerated 2 to 8°C for up to 24 hours. While other factors such as fibrinogen, APCR, TT, reptilase, AT, chromogenic PC, FXII and antiplasmin activity samples can be refrigerated for up to 4 hours. Extreme temperatures should be avoided.²

The delay before whole blood processing and centrifugation depends on the coagulation assay with the most stringent being UFH samples, it should not exceed one hour. This is due to the possible heparin neutralization by platelet factor 4. Testing should occur within four hours.² Short-Term storage and stability of centrifuged whole blood specimens for coagulation testing depends on the coagulation assay being performed. Specimens should be stored capped and sitting for less than 30 minutes, if uncapped there will be an increase in pH, maintaining the pH is important in coagulation testing. An increase in pH can lead to clinically significant prolongation of the PT and aPTT.²

Samples for PT testing can be stored in an unopened tube with the plasma on the cells at RT for 24 hours as well as fibrinogen and D-dimer. The aPTT however is stable for 8 hours under these conditions, however this may not be optimal for abnormal samples. A shorter time should be used and up to 8 hours can be used for exceptions.²

Studies looked at long term stability for PT, aPTT, fibrinogen activity and antigen on normal plasma samples stability (−20°C and −70°C). PT and APTT were significantly prolonged after freezing at both −20°C but showed < 10% change for up to three months at −70°C. There was little to no effect on fibrinogen activity and antigen for up to four months.2 The recommendation for frozen storage of coagulation samples is up to three months at ≤ −20°C and up to 18 months at ≤ −70°C. These studies consider the shortest analyte stability time (ie, Factor VIII and APTT) that yields less than 10% degradation from fresh plasma values.²

ADDITIONAL STUDIES:

Samples from offsite laboratories or clinics struggle with the delivery time of samples and it often exceeds the recommendations of the CLSI guidelines. It recommends that if such samples cannot be analyzed within the recommended time of 24 h the plasma should be removed from the cells, without disturbing the sedimented cells and frozen at −20 ℃ or below for short-term storage (up to 2 weeks) and −70 ℃ or below for long-term storage.¹

There are no recommendations that define acceptable changes when compared with reference values.3 Clinically relevant differences in these studies were defined as a mean change of >10%, and changes of >10% in individual samples. Samples may be statistically significant; however it is important to demonstrate clinical significance.¹

One study looked at the stability of coagulation assays in citrated whole blood transported at ambient temperature up to 2 days after specimen collection which may occur when using a reference laboratory. Five specimens were collected from a single patient and were batched and frozen. An initial assessment looked at the effect of freezing and thawing. Results showed that there was a statistically significant difference in the aPTT (fresh 30.6 sec, thawed 33.0 sec.). Other analytes such as PT, fibrinogen, Factor II (FII), Factor VII (FVII), Factor X (FX) and the D-dimer were not impacted by the freeze thawing procedure.³

It was shown that the PT and non-heparin aPTT can be performed 24-28h after blood collection without clinically significant changes. These findings support previous studies. In patients receiving vitamin K antagonists the INR values are stable up to 24 hours.³

The study also demonstrated all other clotting factors including vWF, AT, PC, and free PS can be tested up to 48-52 hours after venipuncture without showing clinically significant changes and were below the 10% initial measurement. D-dimer also showed excellent agreement after 24 hours at RT. Testing can be safely performed in whole blood samples transported up to 48—52 h. The changes of PT and aPTT results that were clinically significant resulting from decreased levels of FVIII and FV at 24 hours and FVII at 48 hours.³

One study looked at factor activity of FII, FV, FVII, FX, FXII and FXII in citrate-anticoagulated plasma stored for 0 (baseline), 2, 4, 6, 8, 12, and 24 h at 4 and 25 °C, and compared the factor activity differences for various freeze-thaw times (snap freezing at −80 °C and thawing in a 37 °C water bath) It was found that FII, FV, FVII, FX, FXI, and FXII all gradually reduced with storage time at both 4 and 25 °C. Among the factors, FV:C reduced the fastest and the change reached −60% after storage for 24 h at 25 °C. The mean percent changes in the factor assay results were less than 10% compared with the factor assay results at baseline values after storage for 8 h at 4 °C and 4 h at 25 °C. When the storage time was prolonged, the acceptable times for stability of FII, FV, FVII, FX, FXI, and FXII were 24, 8, 8, 24, 12, and 12 h at 4 °C and 24, 4, 8, 8, 12 and 12 h at 25 °C. Among the factors, FV:C was the most unstable, and the stability of FV and FX at 4 °C was better than that at 25 °C.¹

In evaluating the stability of coagulation factor activities in fresh citrated- plasma found that samples for FVIII and FIX C can be safely stored for ≤2 and ≤4 h at both 4 and 25 °C.¹

OTHER CONSIDERATIONS:

A good quality venipuncture and timely centrifugation is important and extremely important in borderline and abnormal results. This was seen in low normal AT results and FVIII levels. A rapid change in FVIII was noted when PPP (platelet poor plasma) was left at RT which is important in vWD diagnosis as well as evaluating for elevated levels relative to recurrent VTE, resulting in lower levels and mis diagnosis. As a result, when PPP is prepared within 24-48 hours FVIII, FV, AT results will be impacted. If you are performing multiple testing on a sample, the shortest time for stability must be adhered. The time delay between blood collection, PPP-preparation and sample freezing must be strictly defined.3 It is important to use a non-frost-free freezer, since frost free freezers have warming of the walls which can expose plasma to freeze and thaw cycles.²

Care should be taken with samples transported on dry ice since this can cause an acidification and variation of several coagulation parameters including PC and PS. Samples from healthy volunteers showed that prolonged storage (16 hours) on dry ice showed a drastic decrease of PC chromogenic levels as well as an increase of PT/aPTT and a decrease in fibrinogen levels. Studies also showed statistically significant variations of LA screen and confirm, FX, D-dimer. Other parameters were not clinically significantly affected.⁴

Other things to consider are type of tube (polypropylene tubes with or without screw caps as well as how samples are thawed (opened or closed). A decrease in pH after transport on dry ice can cause a significant increase of DRVVT, silica clotting time impacting interpretation and possible false positives. This effect may be minimized by placing samples at -70°C for 96 hours before thawing or by thawing open tubes.⁴

There is also limited information regarding multiple cycles of freezing and thawing . One study demonstrated no difference in FV, FIX, FXI, FXII, FXIII, AT activity, PC activity, PS activity, plasminogen activity, UFH anti-Xa activity, and vWF:RCo assessment results. Minor statistical significance, however not clinically relevant for PT, aPTT, fibrinogen, FII, FX and DRVVT. DOAC testing appears to be stable for up to 3 cycles of freeze thaw.⁴ Results of another study showed that the baseline results and all freeze-thaw results for FII, FV, FVII, FX, FXI, and FXII with changes of >10% in each the thaw cycles are 2, 2, 3, 3, 2, and 1, respectively.¹

CONCLUSIONS:

It is important to make sure that when looking at published studies the patients/samples studied should represent the same patient population that you are testing. It should also represent patients who are on oral anticoagulants, hepatitis, liver disease as well as acquired and congenital coagulation disorders. Also, manufacturers' recommendations should be adhered to, there may be additional impact with reagents and instrumentation.

An important consideration is that since multiple coagulation parameters can be evaluated on the same sample the delay and transportation must be adapted to the least stable analyte. Time to report emergent samples must able be agreed upon among institutions, clinicians and laboratory professionals.

Utilizing best practices relating to stability will ensure your coagulation results reflect the coagulation profile of your patients.

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REFERENCES

1. Ying Zhao1, Guofang Feng2, Limin Feng , Effects of pre-analytical storage time, temperature, and freeze-thaw times on coagulation factors activities in citrate-anticoagulated plasma, Ann Transl Med 2018;6(23):456
2. CLSI. Collection, Transport, and Processing of Blood Specimens for Testing Plasma-Based Coagulation Assays. 6th ed. CLSI guideline H21. Clinical and Laboratory Standards Institute; 2024.
3. Manuel Zürcher, Irmela Sulzer, Gabriela Barizzi, Bernhard Lämmle, Lorenzo Alberio, , Stability of coagulation assays performed in plasma from citrated whole blood transported at ambient temperature, Thromb Haemost 2008; 99: 416-426.
4. Claire Flaujac,PharmD1 Céline Delassasseigne,PharmD2 Marie-Françoise Hurta ud-Roux,MD3 Benedicte Delahousse, PharmD4 Elodie Boissier,PharmD5 Céline Desconclois,PharmD6 and the Working Group on Preanalytics in the French Society of Thrombosis Haemostasis, Stability of Hemostasis Parameters in WholeBlood, Plasma,and Frozen Plasma:Literature Review and Recommendations of the SFTH (French Society of Thrombosis and Haemostasis) Seminars in Thrombosis and Hemostasis, August 2024.

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