Refrigeration has long been a critical component of myriad clinical and biological research applications. Nowhere is the need for precision refrigeration and freezing in greater demand than in laboratories that handle blood, blood products, DNA, enzymes, and tissue samples, and in those involved in human reproductive technology.
CRYOGENIC FREEZER: Revco Scientific’s Ultra-Low freezer can chill to -86°C
To enable pre-servation at a rate specific to the mass and cellular type of any given tissue, biomedical scientists are brainstorming with refrigeration engineers on new ways to control the cooling curve to achieve what is known as controlled-rate cooling. It is in this area that much of the new technology is coming into play.
On the whole, Brothman underscores, it is virtually impossible to conduct work in the life sciences without some form of equipment to refrigerate or freeze. Because of this, manufacturers are racing to keep pace with scientists' demands.
Refrigerators that operate at about 4°C and freezers capable of plunging temperatures anywhere from -18¡C to an ultra-low -86°C, such as Asheville, N.C.-based Revco Scientific's Ultra-Low freezers, are among the workhorses in biomedical laboratories. In addition to these systems are a new breed of icy superchillers called cryogenic freezers, such as the Cryostar Cryogenic from Harris Manufacturing Co. of Asheville, N.C. Harris is finding new markets for mechanical units that drop the temperature to -150°C. These units are creating new competition for liquid nitrogen storage units, the traditional cryopreservers that have been able to hold bone marrow, human embryos, or sperm, for example, in suspended animation at -196°C.
Fitting the appropriate cooling system to a specific laboratory need is as crucial as the design of an experiment, engineers and biomedical scientists observe. "Without precise refrigeration or freezing, everything is lost," says Tom LeClair, vice president of scientific sales at Kelvinator Scientific, a division of the Frigidaire Co. in Conway, Ark. Refrigerating and freezing, he explains, is basically the battle of keeping heat at bay-and that means equipment must keep the temperature where it is required at all times.
| Forma Scientific Inc.|
Harris Manufacturing Co.
Jewett Refrigeration Co. Inc.
Minnesota Valley Engineering
Southland Cryogenics Inc.
"We had a big problem with one of our ultra-low [temperature] storage freezers," recalls Rubinstein, who also heads the center's Placental Blood Bank. "Every time the door was opened, the temperature would increase." He turned to ITC Thermodyne, a Sacramento, Calif.-based refrigeration research and development company that custom-designs freezers by retrofitting existing ones. Rubinstein sees retrofitting as a way to save money and keep ambient heat from destroying specimens.
"We've been discussing a way to develop a small vestibule in the freezer with a small door, so that his samples can be inserted without opening the freezer's main door," explains Cody Cadinha, ITC Thermodyne's president and chief engineer. Cadinha believes not only that such a chamber can be developed, but also that he can accommodate Rubinstein's need for a robotic system inside the freezer. Once a technician has placed the specimen in the vestibule, he says, a robotic device can move it to an assigned area on a storage shelf.
Controlled-rate freezing, Rubinstein's other concern, is a matter that has sent many companies to the drawing board, notes Southland Cryogenics' Brothman. "The idea is to freeze at a half-degree per minute or 1 degree per minute, and it will take a very sophisticated device that will use a microprocessor or computer to control a variety of parameters of the freezing curve."
Cadinha is working with Rubinstein on a small, box-like device that can achieve controlled-rate freezing. "All of this is [controlled] through a microprocessor's logic," explains Cadinha. "There can be a program for red blood cells, white blood cells, stem cells, whatever."
When the optimum temperature is achieved in a controlled-rate freezer, "you move [the specimen] to a liquid nitrogen unit," Brothman says.
Inventiveness is vital, scientists maintain, especially when it comes to getting specimens cold and maintaining the temperature. "You have to be creative, otherwise whatever you're trying to preserve will die," contends cryobiologist Joseph Feldshuh, medical director of Idant Laboratories in New York. Idant was one of the United States' first commercial sperm banks.
STORAGE SYSTEM: Minnesota Valley Engineering’s carousel system for liquid nitrogen tanks prevents heat-infiltration problems caused by stacked-rack systems.
In addition to precision temperature control, manufacturers of refrigerators and freezers are continually updating their monitoring and alarm systems so that users have more accurate information about temperature fluctuations-and are warned should something go awry. "The only way to know that you've got continuity of temperature is to have a monitoring device that shows what's going on in the unit at all times," says Tom Reider, vice president for sales and marketing at the Jewett Refrigerator Co. Inc. Like Kelvinator, Buffalo, N.Y.-based Jewett manufactures refrigerators and freezers in the 4°C range to about -86°C. Kelvinator's Laboratory and Pharmacy refrigerators (4C) are widely used in the U.S. and in other countries. Jewett also has a full line of refrigerating systems in that temperature range, such as blood-bank refrigerators and chromatography cooling systems. Both companies' refrigeration units come equipped with monitors and alarms.
HIGH RATE OF RETURN: Jewett’s blood bank refrigeration systems operate from 4°C to -30°C
Monitoring devices are part of the built-in safeguards to keep users aware of temperature continuity and fluctuations, explains Kelvinator's LeClair. The recorded data allow users to compare temperature precision over days, weeks, months, or even years. Other safeguards in both refrigerators and freezers are backup generators that switch on in the event of a power failure.
"All of this is extremely important because of what is stored inside the units," notes Jim Covault, product manager for cryopreservation at Forma Scientific Inc. in Marietta, Ohio. In many cases, he says, storage involves life-support substances or rare tissue samples.
Indeed, at the Mayo Clinic's Lymphoma Tissue Bank in Rochester, Minn., scientists have amassed what they say is the world's largest collection of lymphoma tissue samples. An estimated 10,000 specimens from around the world are stored in two Kelvinator Ultra Cold freezers. Unlike Rubinstein, who is interested in controlled-rate freezing, Barbara Crawford "snap freezes" samples that arrive in Rochester packed in dry ice at about -20°C. "At this temperature, " says Crawford, who supervises the staining laboratory, "[a specimen] will hold for a few days, say, if it comes from as far away as France. We store at -70°C, and at this temperature it can be kept indefinitely."
The collection, in fact, has been maintained since the early 1980s, notes Crawford. The freezers are equipped with electronic seven-day recorders that chart temperature, time, and date. The freezers are very dependable, she reports. A technician reads temperature data daily.
FROM FORMA SCIENTIFIC: The Cryoplus 2 liquid nitrogen unit
"We have a monitoring unit to which you can link 24 pieces of equipment," Covault says. Forma Scientific's 1535 Monitor, for example, can monitor a -86°C freezer, a liquid-nitrogen tank probe, a CO2 incubator, and a circulating water bath. Both real-time and historic temperature data can be obtained. Should any one unit develop considerable temperature fluctuation problems or, worse, lose power, the unit signals an alarm.
An alarm means that heat is in the environment and it's time to panic, explains Michael Pratt, executive vice president of the Central Florida Blood and Tissue Bank. A medical technologist by training, Pratt says blood banks rely on tightly regulated temperature control. His organization uses Jewett's blood bank refrigeration and freezer systems that operate from 4°C to -30°C. Temperature is documented by stylus-and-ink on seven-day chart recorders, much like the data recorders on the Kelvinator freezers at the Mayo Clinic.
"Each blood product that is used in transfusion has its own temperature range for storage," Pratt says. The more thermolabile the cell type, the colder the storage units have to be, he explains. That is why different, highly specialized refrigeration units are used in blood banking. Because plasma contains highly labile components, it must be quick-frozen shortly after being withdrawn from a patient. The optimum temperature for preservation is between -18°C and -30°C. Red blood cells and whole blood can be maintained at significantly lower temperatures, usually in the range of 1°C to 6°C.
Blood and blood products have such rigid storage standards-set by the Food and Drug Administration, the American Red Cross, and the American Association of Blood Banks-that the regulation has driven manufacturing methods of blood-banking refrigeration, Covault explains.
"Anything in the 4°C range to about -30°C operates like a home refrigerator," he says. That means air is fan-blown across a condenser at a constant rate and circulates at all times inside the refrigerator. Scientific refrigeration systems, however, perform this task far more efficiently than home refrigerators do. For blood storage, that means heavier cold air does not settle at the bottom of the unit. If that were to happen, warm air would rise and heat blood on upper shelves.
"Without the fan, cold air would fall on the floor every time the door was opened-and that would allow warm air the opportunity to infiltrate," Covault notes.
Pratt is extremely pleased with his Jewett blood-banking refrigerators and freezers. He says he has no intention of switching to another manufacturer. Jewett also manufactures cold rooms that are used for storage of human remains in anatomic pathology laboratories.
Ultra-low-temperature freezers, meanwhile, induce chilling differently. And knowledge already gained from how to make freezers increasingly colder is spurring new research into both the mechanics and the refrigerants involved in super deep-freezing. Studies of various exotic refrigerants and innovative technologies also could mean the development of mechanical freezers that can go even lower in temperature than -150°C.
"We have a lot of blends now that are used to achieve ultra-cold temperatures," reports Cadinha of ITC Thermodyne. "And it is possible to get even colder." A mechanical freezer, he predicts, ultimately will match the superchilling capabilities achieved now only through liquid nitrogen. "We're just about there now," he says.
Delthia Ricks is a science writer based in Orlando, Fla.