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Specimen Tube “Head Space” and pH

A colleague asked about an issue with “head space” and surface area in sodium citrate (blue-closure) tubes causing changes in pH with the changes becoming more profound the longer the tube is stored. This then affects test results.  My friend had not heard of this before although he knows there are pH changes associated with platelet activation and degranulation that occur in partial draw tubes. Any thoughts and/or papers on this?

Yes, your question provided me with the opportunity to correspond with Drs. Dorothy Adcock-FunkGuiseppe Lippi, and Emmanuel Favaloro, who recently publishedAdcock Funk DM, Lippi G, Favaloro EJ. Quality standards for sample processing, transportation, and storage in hemostasis testing, Semin Thromb Hemostas 2012;38: 576–85. The article is a comprehensive review of hemostasis specimen management issues and is required reading for anyone managing a coagulation laboratory. Dr. Favaloro’s response was, “My understanding from my reading is that pH change has an effect on many hemostasis tests including platelet function and PT/APTT, and is caused by diffusion of carbon dioxide from the plasma into the ambient atmosphere. The larger the air space or caliber tube, the larger the available area for diffusion, and thus the larger feasible pH change. Processed samples are supposedly more susceptible to changes in pH than are whole blood samples due to the loss of the hemoglobin buffering capacity.”

Dr. Adcock-Funk, who provided the article’s section on pH, wrote, “I believe Emmanuel is spot on with his explanation! While I don’t recall a specific paper, this topic is fairly well described in both of Doug Triplett’s books, the one on general coag testing and the one on platelet testing and therefore as you may imagine, both site studies were performed decades ago.” Like most “clotters,” I keep Dr. Triplett’s book, Laboratory Evaluation of Coagulation, 1982, on my shelf. Chapter 10, written by Cathy S. Harms, MT (ASCP) says, “The pH of unstoppered citrated plasma can increase as much as from 7.1 to 8.5 over four hours. Since blood coagulation depends on a series of enzymatic reactions, pH must be controlled to ensure optimal coagulation factor activity. ” Her reference is Zucker S, Cathey MH, West B. Preparation of quality control specimens for coagulation. Am J Clin Pathol 1970;53:924.

Dr. Lippi responds, “Emmanuel’s explanation is indeed almost reasonable. The leading biological “buffer” system in vivo is bicarbonate, followed by phosphate, proteins and hemoglobin. I am not aware of which was the original query, but I do not remember any specific paper addressing the issue of pH changes on coag or platelet testing.”

I also refer you to Thomas Exner’s July 23, 2012 post and Herb Crown’s July 28 comment on the effect of mixing study incubation on lupus anticoagulant testing, which references Exner T. Conceptions and misconceptions in testing for lupus anticoagulants. J Autoimmun 2000;15:179–83. The article and comments address the profound effect of pH in lupus anticoagulant testing, indirectly shedding light on the specimen pH issue.

The relationship of atmospheric exposure and pH with coagulation and platelet activation results are long-established, however I could find no references specifically related to head space, which is the air space above the specimen in a closed tube. It is reasonable to presume, like Dr. Favaloro, that excessive head space relative to specimen, and excessive specimen surface area exposure, even in a sealed tube, will raise the pH. This is one good reason for storing the specimen upright. I’m certain tube manufacturers such as BD Vacutainer and Greiner possess proprietary data on this issue, as well as data on tube wall gas permeability, but are unlikely to publish, given issues of competition. Nevertheless, I have addressed the question to scientists at BD and Greiner in an attempt to collect advanced information, so please watch for more on this subject.

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