Anne D’ Agostino asks, “Should we reject hemolyzed samples from someone in DIC?” The simple answer is “do not reject,” because the hemolysis is likely intravascular, reflecting DIC‘s microangiopathic hemolytic anemia [MAHA].
But to elaborate, there’s no foolproof way to differentiate intravascular [in vivo] hemolysis from spurious [in vitro] hemolysis, the consequence of specimen collection or management error, by laboratory means. Perhaps the most reliable approach is to ensure the specimen is collected by non-traumatic venipuncture and not through a vascular access device. If the “clean” specimen is hemolyzed, you have no choice but to test and report with a comment. A study, Montaruli B, Guiotto C, Cosseddu D. Influence of hemolysis, icterus and lipemia on coagulation tests as performed on Cobas t511 new analyzer. Blood Coagul Fibrinolysis. 2020;31:48-54. doi: 10.1097/MBC.0000000000000873. PMID: 31789660 indicates that moderate hemolysis prolongs the PTT and reduces fibrinogen and antithrombin compared to a non-hemolyzed specimen, but while the variances are statistically significant, they are not clinically significant. Conversely, the authors determined that the D-dimer is both statistically and clinically lowered by free hemoglobin’s optical interference.
One set of investigators has attempted to employ hemolysis corrective formulas to distinguish in vivo from in vitro hemolysis but could make no firm recommendation: Lippi G, Avanzini P, Pavesi F, et al. Studies on in vitro hemolysis and utility of corrective formulas for reporting results on hemolyzed specimens. Biochem Med (Zagreb). 2011;21:297-305. doi: 10.11613/bm.2011.040. PMID: 22420244.
Most automated clinical chemistry analyzers and several automated coagulometers now offer optical serum or plasma hemolysis, icterus, and lipemia [HIL] estimation, enabling the release of valid results based on HIL interference levels upon individual analytes. As reported in the Montaruli study, visible hemolysis is defined as the presence of cell-free plasma hemoglobin at a concentration of over 300 mg/dL, a level that may not indicate optical interference of clot-based assays. However, visible hemolysis may indicate the activation of procoagulants and platelets through venipuncture turbulence or excessive specimen agitation, so unless the hemolysis is intravascular, the results should not be reported.
I’ve requested comments from “the usual suspects,” so watch for additional information.
Dave McGlasson sent a summary of an 2023 ISLH presentation: Tarpley G, Horner K, Bowyer A, Kitchen s.The use of paired matched samples to determine the true effect of haemolysis interference on routine coagulation tests on the Sysmex CN-series coagulation analyser. The investigators identified hemolysis using the Sysmex CS-5100 index and collected non-hemolyzed specimens from the same subjects within four hours of the first specimen. Using hemostasis data from the Sysmex CN-8000 they found that the PT, PTT, and TT were shorter in the hemolyzed specimens. Results didn’t correlate with plasma hemoglobin concentration, but they concluded that hemolysis may influence patient diagnosis and treatment for PTT, PT, fibrinogen, D-dimer, and TT. Though a small study, the results are noteworthy as they are derived from clinically hemolyzed specimens and not those manipulated in vitro.
To accept or not to accept hemolyzed specimens for coagulation testing has long been an issue for the laboratory. I find the best article was Hernaningsih Y, Akualing JS. The effects of hemolysis on plasma prothrombin time and activated partial thromboplastin time tests using the photo-optical method. Medicine (Baltimore). 2017 Sep;96:e7976. This article makes a very simple case. They concluded that even though the results of hemolyzed samples may be statistically different, the differences are not clinically relevant. Therefore, the authors state that not all hemolyzed samples should be rejected for PT and APTT-based tests using photo-optical methods.
I do however state that in personal experience when performing platelet functional testing specimens using platelet-rich plasma or whole blood, hemolysis did play a factor in the testing outcomes. I did note that platelet function was activated in hemolyzed specimens in Chrono-Log, PFA-100, PFA-200, and Multiplate platelet functional analyzers. I didn’t publish this work because it was proprietary at the time I was running these protocols. I would make a smear on these specimens and see micro-thrombi that would affect the testing.
Thanks so much for all of your answers. I did not see the specimen but apparently the specimen was “essentially whole blood and was continuing to clot in the tube“ so they concluded it wasn’t appropriate for testing as the D-dimer would not have given them any more useful information. I’ve never seen a specimen like this one and I just wanted to clarify that coags should not be run. I’m not sure I fully understand the decision but I’ll mull it over some more and of course, will follow laboratory protocol.
Thanx EJF, for sure most of the time it’s a collection error, those few times it is not really stick in my mind.
My first go was a complaint about the hemostasis lab rejecting x3 a citrated sample from a neonate. I went up to see how they were collecting the sample, and all they were doing was allowing the blood to drip from an IV line into the tube, so there was absolutely no possibility of in-vitro hemolysis. The Chem sample was hemolyzed with a normal K+, so I asked them to run LDH and it was outside the EMI; tada! not ex-vivo. Subsequently, (at least before pulling the plug), I often strolled over the Chem (or called) to inquire about their sample appearance before I initiated a “let’s get another one” call. BG
From Professor Giuseppe Lippi, University Hospital of Verona via Email: We measure haptoglobin (even stat) in such cases, which is very helpful. Haptoglobin is reduced in intravascular hemolysis, but almost normal in in vitro hemolysis. See https://www.aacc.org/cln/articles/2018/december/distinguishing-in-vivo-versus-in-vitro-hemolysis#:~:text=Decreased%20concentrations%20of%20haptoglobin%20in,haptoglobin%20is%20usually%20not%20affected.
Best, Giuseppe
Response from Dr. Favaloro
Thanks, BG, this illustrates the benefit of thinking outside the specialisation of hemostasis to get the required answers; we came up with similar thoughts to evaluate right or wrong sample received in: Lippi G, Salvagno GL, Adcock DM, Gelati M, Guidi GC, Favaloro EJ. Right or wrong sample received for coagulation testing? Tentative algorithms for detection of an incorrect type of sample. Int J Lab Hematol. 2010;32:132-8. doi:10.1111/j.1751-553X.2009.01142.x.
my suggestion for repeat samples is to cover the lab, especially when clinical info is not available. I agree this would be problematic for pediatric/neonate; as an adult hospital, pediatric/neonate samples are rare events at Westmead.
From Robert Gosselin via email: GF et al, I would not necessarily use repeat testing to differentiate in-vivo from ex-vivo hemolysis, especially if there is a critical issue (cited DIC). With in-vivo hemolysis, you can document wildly increased LDH with normal potassium In ex-vivo hemolysis, there will be normal LDH with wildly increased potassium. I always check the chemistry sample for the same appearance and look at their data. My history includes multiple draw requests for such an issue in a neonate with in-vivo hemolysis, and same with ETOH and MOH exposure.BG
I think George has captured the essence of the situation correctly. It is difficult for the lab to know if a hemolysed sample received is due to in vivo hemolysis or ex vivo, unless adequate clinical information is provided. Cancelling tests because of hemolysis is problematic, since if the hemolysis is in vivo, getting a new sample will not fix the problem, and continued test cancellation will not benefit the patient, and will frustrate and anger requesting clinicians. Thus, it is probably safer to do the tests requested and flag the sample as hemolysed, requesting a repeat sample for confirmation. Most modern hemostasis instruments can ‘overcome’ the hemolysis by switching wavelengths for clot detection. This will then provide an ‘accurate’ test result reflective of the sample presented to the lab; however, it may not be an ‘accurate’ representation of the patient’s status – especially if the hemolysis was due to an ex vivo event. Hemolysis is an indication of cell activation/red cell destruction, which is typically accompanied by release of coagulation activators.