Improved treatment of frozen red blood cells by red blood cell processing technology / 军事医学

Objective To improve the buffer for washing frozen red blood cells(RBCs)and explore the feasibility of replacing the middle buffer of 2% NaCl with 0.9% NaCl solution in the protocol of deglycerolization and the effect was evaluated.Methods Two units of RBCs separated from whole blood of healthy donors were frozen at -80℃.The thawed frozen RBCs were treated with a washing machine.On the basis of the old protocol(protocol 1),the middle buffer of 2%NaCl was replaced,and the usage of washing buffer and the washing steps were adjusted to create a new protocol(protocol 2).The quality of RBCs washed by the two protocols was evaluated.Results According to the results of the detection of RBCs treated by protocol 1 and 2: the hemoglobin contents(g)were 42.18 ±3.35 and 44.98 ±1.68,respectively,free hemoglobin contents(g/L)were 0.53 ±0.06 and 0.45 ±0.05 respectively, the residual amount of white blood cells (×107)was 1.92 ±1.04 and 1.12 ±1.12,and the osmolarities(mOsm)were 327.0 ±9.06 and 331.8 ±10.62 respectively. Sterile experiments were negative for both bacteria and fungi.The hemolysis rates(%)were 12.02 ±5.78 and 14.30 ± 5.67 respectively.The deforming abilities(%)were 21.42 ±1.45 and 21.32 ±0.84 respectively.The RBC recoveries (%)were 77.18 ±5.58 and 79.63 ±2.06 respectively.The processing time(min)was 79.60 ±0.55 and 78.80 ±1.30 respectively.There was no significant difference in the quality of frozen RBCs washed by the old protocol(protocol 1)and the new protocol(protocol 2)(P>0.05).Conclusion The quality of frozen RBCs washed by the two protocols meet national standards.

Protocol of automatically processing 2 U frozen red blood cells / 军事医学

Objective To improve the processing protocol of the frozen erythrocyte ( RBC) washing machine of the dialysis type to enhance its efficiency of treating the 2 U frozen RBCs .Methods The frozen RBC washing machine for dialysis was used to deglycerolize the 2 U frozen RBCs after thawing .Measures including adjustment of the Pump speed , washing buffer adding and solution osmolarity were taken to decrease the processing time .Hemoglobin(Hb) content, Hb recovery, residual glycerol, free Hb content, deforming ability, and apoptosis of the deglycerolized RBCs were assessed to evaluate the efficiency of the improved protocol .Results As for the cells processed respectively by the previous and improved protocols, contents of Hb were (35.03 ±4.20) g and (53.82 ±2.08) g, respectively; Hb recoveries were (61.10 ±8.46)% and (80.22 ±3.73)%,respectively.Osmolarities representing residual glycerol were (307.00 ± 14.50)mOsm and (322.00 ±29.00)mOsm.Free Hb values were (0.74 ±0.04) and (0.80 ±0.08)g/L.The processing time was (1.64 ±0.31) and (1.04 ±0.16) h,respectively.There were significant differences in Hb content , Hb recovery, and deforming ability between the cells processed by the two protocols respectively .However , there were no significant changes in glycerol contents , free Hb, and apoptosis extent between the cells processed by the two protocols .Conclusion The innovated protocol can improve the quality of washed frozen RBCs , decrease the processing time and enhance the efficiency of the machine.

Usefulness of Frozen-thawed-deglycerolized Red Blood Cells as Quality Control Materials for Red Blood Cell Deformability Test / 대한진단검사의학회지

BACKGROUND: The red blood cell (RBC) deformability test is a useful method for measuring the ability of RBCs to adapt their shape to the flow conditions. Using this test, several investigators have shown the relationship between RBC deformability and numerous clinical conditions. For the quality control (QC) of RBC deformability test, we evaluated whether frozen-thawed-deglycerolized RBCs can be used as QC materials. METHODS: Packed RBCs were frozen with 40% (wt/vol) glycerol and stored at -80degrees C for 3 months. For 10 different frozen RBC panels, RBCs were thawed, deglycerolized and stored at 4degrees C for 4 weeks. Using microfluidic ektacytometer, we measured RBC deformability of the thawed RBCs. The stability of thawed RBCs was tested once a day for 28 days of storage time and was analyzed by simple regression analysis. The precision of the test using thawed RBCs was analyzed for 7 days of storage time by calculation of CV values of intra-assay (10 measurements/assay) and between-day measurements. RESULTS: Frozen-thawed-deglycerolized RBCs were stable for 1 week. Within-run and between-day precisions of the RBC deformability test during 7 days of storage of thawed RBCs were 1.4-2.9%, and 1.9-2.8%, respectively. CONCLUSIONS: Frozen-thawed-deglycerolized RBCs used in RBC deformability test showed satisfactory within-run and between-run precisions and stability for one week after thawing, and may be used as QC materials for this test.

Cryopreservation and Thawing of Red Blood Cells Using Haemonetics ACP 215 / 대한진단검사의학회지

BACKGROUND: The FDA has approved the storage of frozen red blood cells (RBCs) at -80degrees C for 10 years. After deglycerolization, the RBCs can be stored at 4degrees C for no more than 24 hours, because open systems are currently being used. We evaluated Haemonetics ACP 215, an automated, functionally closed system, for both the glycerolization and deglycerolization processes. METHODS: Thirty packed RBCs that had been glycerolized and stored at -80degrees C for 2 weeks were thawed, deglycerolized and resuspended in AS-3. The RBCs were then stored at 4degrees C for 2 weeks. For the evaluation of the procedure, RBC recovery rate, osmolarity, specific gravity, LDH, K+, Hb-2, 3 DPG, Hb-ATP, and plasma hemoglobin were tested at day 0 and day 14. RESULTS: The recovery rate of RBCs was 83.7+/-2.6% (78.9-88.8%). The Hb ATP and 2, 3-DPG of RBCs were 5.16+/-1.0 mol/g Hb and 10.4+/-2.4 mol/g Hb, respectively, at day 0. The supernatant K+, specific gravity, osmolarity, LDH were 1.3+/-0.6 mmol/L, 1.008+/-0.001, 295.0+/-3.1 mOsm/kgH2O, 175.0+/-39.0 unit/L, respectively. All measurements were acceptable to allow the RBCs deglycerolized on ACP 215 to be stored at 4degrees C for 14 days. The blood cultures were negative at day 0 and day 14. CONCLUSIONS: Haemonetics ACP 215 provides a closed, automated system for RBC glycerolization and deglycerolization. This study showed that the RBCs that were glycerolized and deglycerolized in the automated instrument and stored in AS-3 at 4degrees C for 14 days are of an acceptable quality.

The Cryopreservation and Thawing of Red Blood Cells Using 25% Hydroxyethyl / 대한진단검사의학회지

BACKGROUND: The method using hydroxyethyl starch (HES) as a cryoprotectant is a simple, quick, and inexpensive way to make frozen red blood cells (RBCs) for an extended period. It needs neither sophisticated equipment nor skilled labor. As HES is a plasma expander, it does not have to be washed out before transfusion. But, it is not yet introduced in Korea to make frozen RBCs with HES. The aim of our study was to establish a method to cryopreserve RBCs using HES in Korea. METHODS: We rejuvenated RBCs (20 units) on the first day after expiration. They were washed three times with saline adenine glucose mannitol phosphate buffer solution (SAGMP), then their hemat-ocrits were adjusted up to 80%. The same weight of 25% HES was added to each RBC, to make a final mixture of 12.5% (wt/wt) HES-40% RBC. The blood bags were placed in a thin metal frame and frozen in liquid nitrogen. After 3 months, the blood bags were thawed in a 37 degrees Cwater bath. To evaluate the qualities of frozen-thawed RBCs, samples of HES-RBC mixtures before freezing and after thawing were tested for ATP, 2, 3-DPG, potassium, plasma hemoglobin, hemolysis %, and 30-minute saline stability (30-min SS). RESULTS: After thawing, the percentage (mean+/-SD) of RBC hemolysis was 1.6+/-0.4% (range, 1.1-2.6) and three units were over 2%; 30-min SS was 92.0+/-0.16% (88.4-93.8). Supernatant potassium and free hemoglobin were 15.6+/-3.5 mmol/L (10.9-23.0) and 215+/-62 mg/dL (139-318), respectively. The ATP and 2, 3-DPG of RBCs were 4.23+/-0.81 (3.31-6.51) micromol/g Hb and 8.46+/-1.62 (5.21-11.00) micromol/g Hb, respectively. CONCLUSIONS: Of the 20 units of RBCs tested, 17 units (85%) were within the criteria for transfusion (hemolysis88%, potassium<75 mmol/L) after thawing. We could successfully freeze and thaw RBCs utilizing 25% HES as a cryoprotectant.

The Preparation of Frozen Red Blood Cells and Deglycerolization of Frozen RBCs with Haemonetics V50plus / 대한수혈학회지

BACKGROUND: The method utilizing high concentration glycerol is a common way to make frozen red bool cells (RBCs) for extended period, but it requires special deglycerolization equipment for washing after thawing. For economics reason, we could not have a cell washer for wahing the frozen RBCs, so attempted to use Haemonetics V50plus that we have had. METHODS: Twelve fresh packed RBCs were cryopreserved with 40% glycerol method. After 3 months, the RBCs were thawed and washed with Haemonetics V50plus. For the evaluation of the procedure, RBC reovery rate, osmolarity, 30 min saline stability (SS), % hemolysis, ATP, 2,3-DPG, LDH, potassium, and plasma hemoglobin were tested at 24hrs after washing. RESULTS: The RBC recovery rate was 82.1 +/- 4.5% (75.8-89.2) and two units of Frozen RBCs were under 80%. The Hb ATP and Hb 2,3-DPG of RBCs were 5.2 +/- 0.6 micro mol/g Hb (3.9-6.0) and 13.0 +/- 2.1 micro mol/g Hb (8.8-15.1). The supernatant osmalrity, potassium, plasma Hb and LDH were 352 +/- 7mosmol/kg H2O (342~367), 0.8 +/- 0.2 mmol/L (0.5~1.2), 0.8 +/- 0.3 mg/dL (0.4~1.1), 352 +/- 7 U/L (342~367). The 30min SS was 98.9 +/- 0.8. CONCLUSION: We could successfully freeze, thaw, and wash the frozen RBCs with Haemonetics V50 plus.

The Preparation of Frozen Red Blood Cells and a Procedure for Deglycerolizing Frozen RBCs using COBE 2991 Blood Cell Processor / 대한수혈학회지

BACKGROUND: The cryopreservation of red blood cells (RBCs) has not ever been applied to the clinical services in Korea. The aim of this study was designed to supply the frozen-thawed RBCs as a routine service through estimation of efficiency and safety after freezing, thawing and washing. METHODS: Fifteen fresh packed RBCs were frozen with 40 percent(wt/vol) glycerol. After frozen storage at -70degrees for at least one month, the RBCs were thawed and washed in the COBE 2991 blood cell processor. We measured the blood cell count, RBC recovery rate, K+, LDH, specific gravity, osmolarity, and the percentage of hemolysis in the supernatant after deglycerolization. Autologous transfusions were done to the four voluntary donors with deglycerolized autologous blood for clinical assessment. RESULTS: The freeze-thaw-wash recovery rate of RBC was 76.8+/-10.0%, which is not enough to pass the AABB standard. But the recovery rate was increased up to 87.0+/-2.1% with the 4 stepwise predilution technique. The supernatant plasma specific gravity, osmolarity, and K+ were 1.006+/-0.001, 292+/-3 mOsm/KgH20, and 1.1+/-0.2mEq/L, respectively. The Hb ATP and 2,3-DPG were 3.6+/-0.8nmol/g and 13.4+/-4.5nmol/g. In simulated study, the free hemoglobin was 2.8+/-1.1mg/dL and 0.4+/-0.2% of total hemoglobin. In four autologous transfusion cases, plasma haptoglobin level was 96.0+/-40.8 mg/dL (reference range 30~200 mg/dL) and urine hemoglobin was not observed after 2~6 hours later after transfusion. CONCLUSION: The results of this study indicated that technical experiences for freezing, thawing and washing were established for clinical use of frozen RBCs in Korea.

Autologous Frozen-thawed Blood Transfusion in the Bone Marrow Donors / 대한수혈학회지

BACKGROUND: The cryopreservation of Red Blood Cells have been proved to have many advantages in western countries. However, in Korea, clinical application of frozen-thawed blood is still in its early stage. We tried autologous frozen-thawed blood transfusion and evaluated the effects and complications. METHODS: Nine units of whole blood were collected from the autologous (n=1) and allogeneic bone marrow donors (n=3) and made packed RBCs by a centrifugation method. We made frozen RBCs using high glycerol method and stored them in -80degrees C freezer for 1 month. Eight units of frozen RBCs were thawed and washed by Cobe spectra (Cobe, USA). Autologous transfusions were done to the patient and donors while the bone marrow collection procedures were in process for bone marrow transplantation and we carefully observed its effects and complications. RESULTS: The mean RBC recovery rate were 89.8% and the supernatant plasma hemoglobin, K+, LD, osmolality, and simulation test were satisfactory to the allowable limit. After transfusion, the hemoglobin value was significantly elevated being comparable to that of the fresh blood. A case showed mild hematuria related to this transfusion but soon it disappeared in a day. CONCLUSION: The autologous frozen-thawed blood transfusions were safe and as much effective as the fresh blood. It would be essential to have skillful techniques in post-thaw washing process.