Here is a question from “Yellis” at Mayo Labs:
George, I am new to this website. I discovered it through an article in the recent MLO. Do you know the particulars about the hexagonal phase confirmation testing done as a reflex for positive lupus anticoagulant (LA) results? How is it performed? I am not familiar with it and our reference lab has recently added it as a reflex. Thank you.
Hello, Yellis, and thank you for your question.
I first want to thank my colleague and friend, Jeanne M. Isabel, MSEd, CLSpH (NCA) of Northern Illinois University for her What’s New in Hemostasis article in the November issue of MLO. The article references Fritsma Factor, and adds a favorable sidebar. Jeanne, I appreciate your comments.
Hexagonal phase phosphatidyl ethanolamine (HPE) is the key anionic phospholipid in Stago’s integrated Sta-Clot LA kit that I reference in my audio module, Lupus Anticoagulant II. It is so named for its molecular conformation, in contrast to linear anionic phospholipid molecules such as phosphatidyl serine.
You first presume LA is present when an LA-sensitive partial thromboplastin time (PTT) assay (such as Stago’s PTT–LA) is prolonged. What makes PTT–LA sensitive to LA is its low phospholipid content relative to standard PTT reagents.
You may next perform a standard mixing study on patient plasma by adding reagent normal plasma and repeating the PTT–LA. If the result fails to correct (remains prolonged), LA is likely. If it corrects, the original prolongation was likely caused by a factor deficiency. This step rules out prolongation due to factor deficiencies, and may be skipped by those employing the Sta-Clot LA integrated kit.
To confirm LA, you now perform the Sta-Clot LA, which employs two tubes. Tube 2provides HPE, tube 1 does not. Reagent normal plasma is added to both tubes (the plasma also helps rule out a factor deficiency) and a PTT is performed on both. The principle is that since LA reacts with phospholipid-dependent proteins, its PTT result is significantly shortened via HPE neutralization in tube 2. The PTTs from both tubes are recorded and compared, and if the tube 2 PTT is shorter than tube 1 by a difference of 8 seconds or more, LA is confirmed.
The test is described in Schjetlein R, Wisloff F. An evaluation of two commercial test procedures for the detection of lupus anticoagulant. Am J Clin Pathol 1995;103:108-11.
Several companies provide competitive integrated kits that compare to Sta-Clot LA, however they use linear phospholipids as their neutralizing reagent. Only Sta-Clot LA uses HPE, as the reagent was developed and patented by Stago. There may be no inherent advantage to HPE compared to linear phospholipids.
The hexagonal phase confirmatory test called the Staclot LA
The hexagonal phase confirmatory test called the Staclot LA does have an advantage over the linear phospholipid testing in picking up specimens for LA testing that have low Factor V levels and inhibitors. In a wet workshop that was performed at the International Antiphospholipid Antibody Conference in San Antonio, TX, a specimen with low levels of FV with a mild inhibitor was given to all participants. In one group the hexagonal phospholipid confirmatory test was used and a second group used the linear phospholipid confirmatory assay. In the group using the Staclot LA test they properly identified a Factor V deficiency with a mild inhibitor. The participants using a linear phospholipid confirmatory test identified the specimen as having the presence of an LA. This is one advantage when using two confirmatory tests might be an advantage.
David L. McGlasson, MS, MLS(ASCP)
At Clarian Patholology Laboratory, we utilize the dRVVT test
At Clarian Patholology Laboratory, we utilize the dRVVT test and the Staclot LA. We have had several cases of negative dRVVT with a positive Staclot LA. We will be adding the IL Silica Clotting Time test soon. The Staclot LA is performed on our ST4 but we do have a protocol for loading it on the TOP.