Judith Graham Pool and the discovery of cryoprecipitate

The history of concentrated factor VIII (FVIII) begins in the early 1940s, when Edwin J. Cohn 1 pioneered fractionation of plasma with various proportions of ethanol. His ‘fraction I’ contained fibrinogen and also FVIII (but methods of assay had not yet been developed) and von Willebrand factor (which had not yet been defined). The utility of fraction I in haemophilia was demonstrated early 2 and modest amounts were used in developed countries throughout the 1950s and 1960s, but its sterile production required a large laboratory. A commercial version became available in the United States as a concentrate of fibrinogen, rich in FVIII; in one measurement 3, the ratio of FVIII to total protein was sevenfold that of native plasma. In 1965, it cost about 17.5 cents (U.S. $ 0.175) per FVIII unit 4. Meanwhile, community blood banks were separating and freezing plasma from whole blood for local use. Blood banks in the United States generally set the price of plasma low, as a by-product of whole blood collection, so it was widely used. The hemostatic efficacy of whole plasma was sub-optimal because only a limited volume could be infused at one time. In the early 1960s, Cutter Laboratories in Berkeley, California, and its scientists were trying to make an improved concentrate of FVIII, with help from northern California ‘clotters’, including Paul M. Aggeler of the University of California at San Francisco and Judith Graham Pool of Stanford University. I had the felicity of being a haematology Fellow in Dr. Aggeler's laboratory from 1962 to 1965, which were heady years in the history of haemophilia treatment. The first FVIII concentrate I ever saw, in 1963, was an experimental, lyophilized product from Cutter Laboratories. We were planning to extract all remaining, very rotten teeth from a malnourished man with severe haemophilia A, to prepare him for dentures. We dreaded the event: a similar patient, who had the same operation a year earlier, was hospitalized for 2 weeks, needing several bags of plasma per day plus occasional red blood cell transfusions. With the precious new concentrate, however, our patient's hemostasis during dental extractions was perfectly normal. Cutter could not produce much of this marvellous material because the yield of concentrated FVIII from plasma was low. Enter Dr. Judith Graham Pool. She had earned her Ph.D. in physiology from the University of Chicago in 1946 and had joined the faculty of Stanford University in 1953, investigating coagulation with particular attention to FVIII. With Jean Robinson, she had developed the ‘two-stage’ assay for FVIII in 1959. In the early 1960s she used that method to assay FVIII in plasma fractions prepared by Cutter's Drs. Albert Pappenhagen and Edward Hershgold. Their starting material was pooled plasma, frozen in large containers, which they thawed cautiously. They knew that the potency of FVIII in plasma dwindled at room temperature, so they were careful not to thaw the plasma completely. But Dr. Pool found very little FVIII in their thawed supernatant. She then tested the small amount of unthawed, fibrous-looking paste at the bottom of the containers, and, in 1964, found the FVIII 5. The genius of Dr. Pool was her leap from that laboratory observation to the practical idea of using the last-to-thaw property of FVIII to separate ‘cryoprecipitate’ from whole plasma in an ordinary blood bank, re-freezing the small volume in its own sterile plastic bag and storing bags in a freezer so that eventually several could be given to a patient at one time 4. Meanwhile, the thawed plasma could be used for extraction of other plasma proteins. The method was so simple, and cost so little for the additional materials, that it was rapidly copied by blood banks around the world. Cryoprecipitate became widely available not only for patients with haemophilia A or von Willebrand disease but also for patients deficient in fibrinogen or factor XIII. It was a great, miraculous gift to the world. Many tens of thousands of patients have benefitted. Meanwhile, more highly concentrated commercial, lyophilized FVIII preparations were developed, using various technologies (often including cryoprecipitation). The first was licensed in the USA at the end of the 1960s and then cost ten cents (U.S.$0.10) per FVIII unit in Los Angeles. We quickly adopted its use and, by 1970, started to introduce our haemophilia patients to self-infusion at home. Our local blood bank had switched from the use of glass bottles for blood collection to plastic bags only in the late 1960s, delaying the development of cryoprecipitate. Furthermore, instead of charging only the incremental costs of the additional processing required to extract cryoprecipitate from plasma, they divided the total cost of blood processing among all components. As a result, cryoprecipitate cost our patients more, per FVIII unit, than did concentrate. Because it required transport as well as storage in the frozen condition, it was also less convenient. In the Los Angeles area, the use of concentrate for haemophilia treatment predominated over that of cryoprecipitate by a large margin. A very different trajectory developed in Seattle, Washington. Its Puget Sound Blood Bank was devoted strongly to the concept of regional self-sufficiency in blood products. It rapidly developed adequate cryoprecipitate production to meet the needs of local patients and stave off importation of commercial lyophilized products. Patients could take part in self-infusion programs by storing cryoprecipitate in home freezers. The isolationist practices in Seattle did not protect its patients from the transmission of endemic viral diseases, such as hepatitis B, which was equally as prevalent (that is, almost universal) in heavily treated Seattle patients as in Los Angeles ones, when tested in the mid-1970s 6. Their strong code of self-sufficiency did protect many of its patients from the transmission of a new viral disease, AIDS, which was epidemic in Los Angeles 7 in the early years but not in Seattle, to Seattle haemophilia patients using cryoprecipitate 8. Cryoprecipitate has been a special boon to less-affluent countries where cost is a critical consideration. One of the higher elements of cost is the extra plastic bag, typically imported, a problem solved in Thailand by going into production of sterile plastic bags for blood collection themselves (A. Chuamsumrit, personal communication). The original recovery of cryoprecipitated FVIII was said to be about 70% of that in fresh frozen plasma 9, but that level was rarely sustained in routine daily production. Various manoeuvres were tried in an attempt to improve the recovery of cryoprecipitated FVIII from plasma. I participated in some of these experiments and concluded that FVIII could be lost at any step if delays were allowed to occur. More even freezing and thawing throughout the plasma could improve the yield, which could be achieved by freezing the plasma like a pancake in an extra-large bag 10. The yield was further optimized by the Australian thaw-siphon method, in which thawed plasma was continuously siphoned off during the thawing process 11. In Canada, the use of heparin as an anticoagulant was demonstrated to improve recovery 12. Although others confirmed that observation, heparin use did not become popular. When plasma was obtained by plasmapheresis of repeat donors, the FVIII content could be improved by choosing donors with high FVIII levels or by increasing donors’ levels with prior administration of DDAVP, a practice that never became popular. In the 1980s in Chicago, however, the father of a boy with haemophilia A underwent plasmapheresis after DDAVP administration on 103 occasions, producing 359,460 IU of FVIII for his son 13. None of these innovations has become a widespread feature of cryoprecipitate production. In the short but critical interval between the beginning of the AIDS outbreak and the availability and application of the HIV-antibody test (first used in March, 1985, at the Irwin Memorial Blood Bank in San Francisco), we could rely only on closer selection of donors. Designated-donor programs proliferated. In Los Angeles, an epidemic city for AIDS, we had a designated-donor plasma program in the mid-1980s but it could serve only a few patients with modest transfusion needs and it was very costly. We used geographical designation on one occasion when a rarely infused young man with mild haemophilia A required a surgical operation. His mother, director of a blood bank in Idaho, a non-epidemic area, shipped us sufficient Idaho cryoprecipitate for his procedure. Once the transmission of AIDS and other viral disorders was recognized, and viral-inactivation techniques applied in the 1980s to plasma-derived concentrates, some blood banks applied viral inactivation methods to cryoprecipitate 14 or the plasma from which it was made 15, 16, albeit with loss of some FVIII and von Willebrand factor potency. Most cryoprecipitate in use in the world today, however, is not viral-inactivated. By the end of the 1990s, Bruce Evatt of the Centers for Disease Control and his colleagues 17 warned countries that still relied on cryoprecipitate that blood screening for transmissible viruses was imperfect and might not detect newly infected donors. Patients who were treated frequently with cryoprecipitate bore a notable risk of acquiring a blood-borne infection, especially in countries with expanding epidemics of HIV infection. Nevertheless, in most of the world, finances set strict limits and cryoprecipitate remains the product patients can hope to afford 18. For these patients, it remains a godsend. Not surprisingly, Judith Pool became a heroine to haemophilia patients around the world. She received a great many honours and found herself venerated. She found all this embarrassing, for the discovery of cryoprecipitate had been serendipitous, and she was a modest person. It was only one of many interests and achievements in her life, and she was only one of many scientists who had contributed to the development of treatment for haemophilia. People need heroes, however, and it was our happy fortune that, in this instance, the object of all the adulation was a worthy person on many grounds. I was lucky enough to be acquainted with her during my haematology Fellowship in San Francisco and to continue the friendship after I joined the haemophilia program at Los Angeles Orthopaedic Hospital in 1966. I remember her as a gentle, gracious lady, a warm friend. She had intellectual curiosity, an analytical mind and good judgment. Her advice was practical and apt. She was a stalwart of the International Committee on Thrombosis and Hemostasis (forerunner of the Society), which decided matters of standardization, notably nomenclature. Dr. Pool was strongly interested in helping young scientists, especially women who, in her era, sometimes had a hard time. In her role as an advisor at the National Institutes of Health, she quietly arranged for the committee that devised a standardized (Bethesda) test for FVIII inhibitors. Working behind the scenes, she had assigned me as chair. Given that, at the time, I had less expertise in inhibitors than several of the committee members, I suspect that her motive was to promote my career. She did not take part in the committee's heated debates but she critiqued my resulting short report. It was published shortly after her death in 1975 of a brain tumour, at the age of 56, much too young. I felt grateful and privileged to have had her encouragement and friendship. The author stated that she had no interests which might be perceived as posing a conflict or bias.