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editorial
. 1998 Mar;36(3):855. doi: 10.1128/jcm.36.3.855-855.1998

Stability of Free and Complexed Human Immunodeficiency Virus p24 Antigens during Storage for Various Intervals

Subhash C Arya 1
PMCID: PMC104648  PMID: 9508335

The multicentric storage of serum specimens that had tested positive for human immunodeficiency virus p24 antigens at 4 and −70°C was associated as well with three cycles of freezing and thawing of these samples. While there was no reduction in the p24 quantum during storage at −70°C, storage at 4°C was associated with a 25 to 27% reduction in free p24 antigen content (1). There was no loss at either temperature when p24 assays were carried out by the immune complex dissociation method. Furthermore, three cycles of freezing and thawing of serum aliquots stored at −70°C had no effect on the p24 quantum.

There are no details in the work of Arens et al. (1) about the rapidity or other factors of thawing and freezing of serum aliquots stored at −70°C. Rapidly thawing specimens stored at −70°C at a room temperature around 22 to 24°C would mean a sudden temperature rise of about 90 to 95°C. In cases when the aliquots are frozen suddenly from room temperature to −70°C, the temperature differential would imply an environment very much like the one encountered during the pasteurization of milk. Such a change could inactivate a fair proportion of p24 antigen. The details of rapidity of freezing and thawing of serum or plasma aliquots stored at −70°C or even at −20°C would have to be defined (1). Only such an analysis would indicate the ideal way to freeze and thaw p24-positive specimens to subzero temperatures.

In all probability, temperatures around 4°C were monitored by conventional usage of thermometers in the previous study (1). Ideally, the monitoring could be done precisely by using electronic temperature loggers. They could be preset to record the temperature half-hourly, and any inadvertent exposure to temperatures less than 0°C could be measured. In Adelaide, Australia, it was possible to monitor the temperatures around vaccines at different storage sites. Retrieval of data from electronic loggers revealed that out of 40 vaccine storage sites, vaccines at 34 sites had been exposed to subzero temperatures for varying intervals, and in 3 sites, the temperature had exceeded 22°C (2).

A comprehensive study of the stability of free and immune-complexed p24 would have to rely on temperature exposures ascertained by electronic loggers (2) and not by conventional thermometers.

REFERENCES

  • 1.Arens M, Meyer III W, Brambilla D, Bremer J, Fiscus S, Griffith B, Hammer S, Hodinka R, Kabat W, Yen-Lieberman B, Reichelderfer P. Stabilities of free and complexed human immunodeficiency virus p24 antigens during short- and long-term storage. J Clin Microbiol. 1997;35:2413–2416. doi: 10.1128/jcm.35.9.2413-2416.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.Warwryk A, Mavromatis C, Gold M. Electronic monitoring of vaccine cold chain in a metropolitan area. Br Med J. 1997;315:518. doi: 10.1136/bmj.315.7107.518. [DOI] [PMC free article] [PubMed] [Google Scholar]
J Clin Microbiol. 1998 Mar;36(3):855.

AUTHOR’S REPLY

Max Arens 1,2, William Meyer III 1,2

In his letter, Dr. Arya expresses concern that the speed at which we froze and thawed the serum may have affected the results by causing the denaturation of some of the p24 antigen present in the specimens. He compares the temperature change of −70 to 20°C experienced by the plasma in our experiments to that of the pasteurization of milk. He also implies that multiple events (i.e., the three cycles of freeze-thaw that we mentioned in our work) would have an adverse effect on the recovery of p24.

In our experiments, the serum samples were frozen quickly by placing the tubes directly into a −70°C freezer. There was no attempt to cool the sera at a predetermined rate. The samples were also thawed rapidly by agitation by hand in a 37°C water bath until the point at which there was no ice remaining and then retained at room temperature prior to the assay (or refrozen, in some cases, for the freeze-thaw experiments). In setting up the protocol, it seemed to us that these procedures would be easily standardized among the several laboratories performing the testing and that they would not be deleterious to the analyte we were attempting to measure.

Our initial experiments (not shown, but referred to in our work) proved that multiple freeze-thaw events did not affect the recovery of p24, since the same quantitative result was obtained on many different samples after one, two, or three freeze-thaws. If the freezing and thawing caused denaturation of the antigen, one would expect that each event would result in incrementally lower values in the quantitative assay. We did not observe this.

The comparison, by Dr. Arya, of our method of freezing and thawing to the conditions of pasteurization of milk is not likely an appropriate one. The temperature differentials among the two processes are similar in magnitude, but the absolute values are vastly different. The suggestion that the temperature differential from −70 to 20°C is as likely to denature proteins as the differential from 20 to 72°C (the temperature achieved in the high temperature-short time method of pasteurization) is not reasonable. A well-established tenet of protein chemistry is that it is the heat, not the temperature range through which proteins pass, that kills proteins.

In addition, in some early experiments we compared the effects of three different thawing temperatures on the recovery of p24 antigen from 18 specimens obtained in our laboratory. The concentration of p24 recovered in samples thawed at 4°C was 187 ± 80 pg/ml, that in samples thawed at room temperature was 202 ± 71 pg/ml, and that in samples thawed at 37°C was 198 ± 102 pg/ml (values are reported as means ± standard deviations). Thus, thawing at these three different temperatures did not affect the average recovery of human immunodeficiency virus type 1 p24 antigen from ultrafrozen replicate plasma aliquots.

We would be happy to discuss other details of our experiments at any time.

Articles from Journal of Clinical Microbiology are provided here courtesy of American Society for Microbiology (ASM)

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