Preservation of human platelets by freezing to −150 °C

ABSTRACTS,

18th ANNUAL

amount of retransfused cells. Patients with leukemia, and cancer of the breast and lung were treated in the described manner. The clinical results so far obtained indicate that the cryopreservation procedure is effective enough to allow an extremely high-dose cytostatic treatment. 47. Cryoprotective Properties of DimerhylsuIfoxidc (Me,SO) and Polyethylene Giycol (PEG) during Freezing of Lymphocytes and Peripheral Stem Ceils. M. W. Sc~Erwe, P. GOBEL, R. SEELIS, Zs. PUSZTAI-MARKOS, AND U. ESSERS (Helm-

ho&-Institut fir BMT an der RWTH Aachen, Goethestr. 27/29, D-5100 Aachen, West Germany). The use of MepSO as a cryoadditive for lymphocytes (Ly) and peripheral stem cells (CFU-c) yields high m covery and reproducibility; on the other hand, toxicity and osmotic activity must be taken into account as damaging factors, since they complicate the cryopreservation procedure. Therefore, the extracellular additive PEG was compared to Me,SO. Because of its weak osmotic activity and nontoxicity, improved recovery rates may be expected. Here, separated Ly and CFU-c were frozen together in small cylindrical samples. A newly developed biofieezer was used to establish linear cooling rates in the range from 1 to 60 Wmin after the freeze plateau; whereas the prefieeze cooling rate and the length of Qe plateau phase were kept constant. After thawing in a shaker water bath, cells were counted, viability was tested, and CFU-c were detected in an agar test system. The results show that the CFU-c optimal cooling rate of 1 Wmin is not changed by PEG; in contrast, the Ly optima1 cooling rate is increased when using PEG. In both cases, the absolute recovery values of Me,SO exceed PEG values, after either first or repeated washing. From these findings it is concluded that Me,SO may not be substituted by PEG. The differences of the optimal cooling rate for Ly and CFU-c lead to a possible interpretation of the localization of CPU-c-capabilities and Ly-viability criteria or immunologicai parameters, respectively, to certain sites of the cells. 48. A Method of Calculation of Optimal Cooling Rates for Cryopreservation of Blood Cells on the Basis of Volume Loss During Freezing. hf. W. SCHEIWE, C. K~RFIER, AND K. WOLLHOVER (Helmholtz-Inst. f. BMT, Goethestr.

27129, 5 I00 Aachen, West Germany). One of the aims of modem crybiology is the determination of suitable cooling rates for a given type of cell. Because of the great number of variabIes involved during freezing, a feasible solution of this problem seems highly desirable. Here, three basic results concerning red blood cells and lymphocytes in the

MEETING

625

NaCl-H,O system have been combined in order to get a first approach for solving the problem: (1) The respective optimal cooling rates for both cell types have been established experimentally (red blood cells: 4900 K/min; lymphocytes: 60 Klmin; (2) the volume loss of individual cells during freezing has been determined by cryomicroscopy for a broad range of cooling rates (2 to 600 Wmin); (3) the thermodynamically predicted volume loss of cells during freezing has been compared with Ihe experimental data, and the hydraulic membrane permeability function (L,) was estimated by fitting the calculated curves to the experimental ones. Using the detectedl, and assuming that at the eutectic temperature no more water efflux out of the cells occurs, a lethal intracellular salt concentration during freezing was determined. These findings allow the theoretical prediction of a suitable cooling rate in the NaCI-H,O system, if L, is known. It is interesting that the detected L, for red blood cells corresponds to the findings of Rich et al. (1968); for white blood cells, L, is deterinined the first time. 49. Freeze-Fraciure and Spin Label Studies of Thermai Shock in Human Erythrocytes. T. TAKAHASHI,J.L.BURTON,M.S.CHO,ANDS.NOJI

(ANRC Blood Services Labs, Bethesda, Maryland). Human erythrocytes which are osmotically stressed or treated by several chemicals are hemolyzed by rapid cooling, a phenomenon known as thermal shock. MOlecutar events in this phenomenon were studied by freeze-fracture and spin label techniques. In the freeze-fracture study, intact erythrocytes equilibrated with 1 M glycerol were suspended in isotonic or hypertonic NaCl solutions containing 1 M glycerol at 37°C. Cells were quenched in liquid Freon directly from 37°C or after they were cooled to 10 or 0°C. Intramembrane particles are largely randomly distributed in cells suspended in isotonic solution, and exposure temperature does not affect that distribution. On the other hand, particles are aggregated in cells suspended in hypertonic solution at all temperatures studied. In the spin label study, cells were loaded with spin-labeled fatty acid. Then the cells were suspended in various concentrations of NaCl at room temperature. Hyperosmotically stressed cells showed little change of overall membrane fluidity (described by 2T,,). When the cells were cooled, a further change was observed. In cells suspended in isotonic solution, a plot of 2T,, against temperature gave an essentially straight line. But in cells suspended in hypertonic SOlution, there was a break in the line at 15°C. (Supported in part by NIH Grant GM 17959 and BRSG NO. 2SO77RR05737.) 50. Preservation -150°C. MILEV, KOVAKI,

of Human Platelets by Freezing to Tsv. TSVETKOV, CH. NIKOLOV, A. L. I'SONEV, N. NLEKSIEV, I. I‘ANAND T. LISICHKOV (Central Problem

626

ABSTRACTS,

18th ANNUAL

Laboratory of Freeze-Drying and Cryobiology, Sofia, Bulgaria). Platelets either were frozen at l”C/min to - lo”, then 1OWmin to -80” or were held at -20°C for 30 min. Both sets were then transferred to liquid nitrogen vapor, Freezing was with 5% Me&O in aluminum foil containers. Storage was for 30 days. Addition of Me,SO without freezing reduced clot retraction from 1.02 -r 0.09 to 0.90 ml and, after freezing and thawing, to 0.58 2 0.9 and 0.62 2 0.09 ml, respectively for programmed and stepwise freezing. Hypertonic shock was assayed by measurement of both TZ% and the angle between the steepest tangent and the abscissa. The Tg% values for platelets before treatment and after addition of Me,SO were 98.5 2 2.01 and 72 2 12..54%, respectively. After freezing the values of Ta for programmed and stepwise freezing were quite close: 34.5 f 13.4 and 31.8 k 12.54%, respectively. Tangent-abscissa angles were 108.4 ? 5.51” for fresh platelets and 122.7 + 9.38” after Me,SO addition. After programmed freezing the value was I47 2 8.68” and 152.8 + 5.53 after stepwise freezing. Differences between platelets frozen by program or stepwise procedure were not statistically significant.

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heart sequestration indices were 4.1 f I.2 and 5.2 + 0.9 on the 1st day and 7.3 f 0.6 and 9.1 2 1.2 on the 10th day. The liver/heart sequestration index was - I.2 f 0.3 and -2.2 f 0.5, 1st day, and I.9 + 0.5 and 4.2 -r0.9, 10th day. Recovery of Rozen piatelets at 24 hr is 79.8% of fresh. Platelet life’span is slightly reduced and increased sequestration in the liver is observed. 52. Therapeutic

Effectiveness

of Platelets

Frozen

at

-196°C. S. SUMIDA (Department of Cardiovascular Surgery, National Fukuoka Central Hospital, Jonai 2-2 Fukuoka, 810 Japan). Platelets frozen in 5% glycerol solution were thawed, deglycerolized, and transfused into 59 patients having thrombocytopenia from many causes: idiopathic thrombocytopenic purpura, leukemia, trauma iatrogenic diseases, disseminated intravascular coagulation and others. The circulating platelet count increased in 45 of 59 cases (74.3%) and the bIeeding time was shortened in 36 of 59 cases (64.4%). No patient complained of any side effects. HLA (A, B , and C loci) matched transfusions of frozen platelets were also performed in IS cases; however, no statistically significant difference between the nonmatched group and the matched group was found.

51. A Cotnpararive Study of the Recovery, Sequestration borh Fresh MILEV, K.

DIMITROV,

Viability and Human Platelets and Preserved at -196°C. A. KARAKOSTOV, CH. NIKOLOV. L. L. ‘I~ONEV, Tsv. TSVETKOV, N.

of Autologous

ALEKSIEV (Central Problem Laboratory of Freeze-Drying and Cryobiology, Sofia, Bulgaria) . Twenty volunteers donated a total of I liter of blood into siliconized glass containers with ACD anticoagulant. Platelet concentrates containing 142 X 108 2 Il.1 x log cells were prepared from each donation. Platelet concentrates from 10 of the donors were tagged with Wr and reinfused. The remaining concentrates were diluted with 20-m] autologous plasma and an equal volume of 5% Me,SO, 5% glucose, and frozen to -9°C at l”C/min, then to -80°C at 10”Cimin in aluminum foil containers. Twenty-four hours after infusion, fresh platelets showed 68 2 7% survival and IO c I days life span. Frozen platelets showed 54.3 f 6.1% 24 hr survival and 8 t 1 days Life span. For control and frozen platelets respectively, the spleen/

TABLE-AB category: Glycerolization: Spin after gtycerol;

1 NO

2 3 4

Recovery (%)

wash

Spin- Discard supernatant 3.5% NaCl (500 ml) 0.9% NaCl(SO0 ml) 0.9% NaCl(500 ml) 81.7 2 1.0 (10)

of for

inven-

iSTRACT 53

NaCl(50D NaCl (500

Yes

No spin ml) ml)

O.% NaCl(500 ml) 87.2 2 4.0 (4)

4

3 solution Yes

No spin 3.5% O.%

of Rapid-Freeze-Preservation Cells: Increased Room

tory. S. SUM~DA (Department of Cardiovascular Surgery, National Fukuoka Central Hospital, Jonai 2-2, Fukuoka, 810 Japan). Two hundred milliliters of 45% gIycerol solution (glycerol 45%, mannitol 3%, sorbitol 3%, and NaCl 0.85%) were added to 303 + 33 ml (n = 25) of packed red blood cells (RBC) and mixed well in the blood collection bag. .The glycerolized RBC were spun at ZOO& for 5 min after which the supematant was discarded. The concentrated glycerolized RBCs were transferred into the blood freezing container and frozen rapidly in liquid nitrogen. After a week at -lWC, frozen RBC were thawed in a +4o”C water bath and deglycerolized with 3.5 and 0.9% NaCl solutions as shown in the table below. The prefreeze concentration of glycerolized RBC did not jeopardize the yields of thawed-deglycerolized RBC. All units of RBC processed in this experiment were transfused clinically without any side effects.

2 200 ml of 45% glycerol Yes - 196°C~----+4OT

Freeze-thaw:

Dcgiyeer&zation 1

53. Simplificarion Red Blood

3.5% 0.9%

No spin

NaCl(300 ml) NaCl(300

ml)

O.% NaCl(300 ml) 86.0

* 1.8 (3)

3.5% 0.9%

NaCI

(250

ml)

NaCl(2SO ml) 0.9% Nacl (250 ml) 86.5 2 1.8 (3)