Preparation of leukocyte-poor erythrocyte concentrates by glycerin treatment and washing

The discontinuous washing procedure of red cell concentrates with saline was modified in order to improve the removal of leucocytes and platelets. It was achieved by glycerolization and deglycerolization without freezing and thawing. The elimination rate of leucocytes amounted to 99.9% and of platelets to 97.4% related to whole blood. A unit of red cell concentrate prepared from 400 ml of donated blood contained a total of (0.001 +/- 0.003) X 10(9) leucocytes and (2.35 +/- 1.33) X 10(9) platelets. The relatively high elimination rates are comparable with those obtained by filtration and cryopreservation, respectively. The whole procedure performed in glass bottles required one hour more than washing three times without glycerolization. A further improvement of the procedure should be directed towards saving time in processing and towards lowering the loss of red cells that is still 20%.     

The oxygen transport function of the blood and of the erythrocyte concentrate during storage and its importance for blood transfusion

The property of oxygen transport function was investigated in blood which had been stored in solutions of "glugizir" and "zitroglucophosphate" and in erythrocyte concentrates gained from it on the noughth, 7th, 14th und 21st day of storage at -4 +/- 2 degrees C. The parameters of the oxygen binding function (oxygen content of erythrocytes, half time of haemoglobin saturation with oxygen, concentration of organic phosphate [2.3 diphosphoglycerate and adenosine triphosphate] and those of inorganic phosphorus were determined in erythrocytes. During storage for more than 21 days no significant differences could be detected in the property of oxygen transfer between erythrocytes of stored blood and erythrocyte concentrate, with the values for storing in zitroglucophosphate being somewhat higher. Problems of applying all components of donor blood efficiently are discussed. In performing an adequate haemotherapy with blood components the importance of a functional condition of erythrocytes and oxygen balance in the organism of the receiver should be considered. The necessity of transfusing erythrocyte concentrate in the therapy of anaemias of different genesis is emphasized and the differences in applying concentrates in different plasma solutions are referred to. By transfusing concentrates the effectiveness of hemotherapy are elevated and the rate of complications and side-effects of whole blood are diminished.     

Erythrocyte Concentrates Recovered from Under-Collected Whole Blood: Experimental and Clinical Results

BackgroundAlthough periodic blood shortages are widespread in major Chinese cities, approximately 1x10⁵ U of whole blood are discarded yearly because of under-collection. To reduce the wastage of acid citrate dextrose solution B (ACD-B) anticoagulated under-collected whole blood (UC-WB), this study was performed to elucidate the effect of extracellular pH and holding time on erythrocyte quality. Mannitol-adenine-phosphate (MAP) erythrocyte concentrates (UC-RBCs) were prepared with UC-WB to assess the safety and efficacy of this component.MethodsThe effect of the different extracellular pH levels and storage times on erythrocytes was assessed by fluorescent probes, SDS-PAGE electrophoresis, electron microscopy and spectroscopy. In vitro properties of 34 UC-RBCs that were prepared with UC-WB at different times after collection were analyzed and compared to normal RBCs during 35 days of storage. The results of transfusion with UC-RBCs and the incidence of adverse reactions in 49 patients were determined.Results1) Low extracellular pH levels and long storage time induced increases in RBC fluorescence polarization and mean microviscosity, changes in membrane fluidity, band 1, 2 and 3 protein expression, and erythrocyte morphology. 2) During storage for 35 days, difference in between-subjects effects of K⁺, hemolysis and supernatant erythrocyte membrane protein (EMP) were statistically significant (P = 0.041, 0.007 and 0.002, respectively), while the differences between these parameters in the 4 h group and comparable controls were less significant. 3) Clinical data from 49 patients confirmed that transfusions with UC-RBCs were satisfactory with no adverse reactions.ConclusionThese results suggest that it is feasible to prepare RBCs with ACD-B anticoagulated UC-WB at a minimum of 66% volume of the labeled collection. It was effective and safe to transfuse the UC-RBCs prepared within 4 h after collection and stored within 7 days. The use of UC-WB would be a welcome addition to limited blood resources in China.

Trial Registration

Chinese Clinical Trial Registry ChiCTR-TRC-13003967     

Survival in vivo of platelets stored for 48 hours in the buffycoat at 4 degrees C compared to platelet rich plasma stored at 22 degrees C

High speed centrifugation allows separation of whole blood into cell free plasma, a buffy coat and leukocyte poor red cells. The buffy coat can be used for the preparation of platelet concentrates. High lactate production at 22 degrees C requires storage of the buffy coat at 4 degrees C. Survival in vivo of platelet concentrates prepared from buffy coats stored at 4 degrees C for 48 h (BC-PC) was compared with the survival in vivo of platelet concentrates from platelet rich plasma stored at 22 degrees C for 48 h (PRP-PC). Both methods were studied in the same healthy volunteers (n = 8) using 51Cr labeled autologous platelets. The mean +/- SD recovery 15 min after reinfusion of the BC-PC was 30.5% +/- 13.3% and for PRP-PC 41.4% +/- 7.9% (p less than 0.0001). The survival in vivo for BC-PC was 2.4 days +/- 0.4 days and for PRP-PC 7.0 days +/- 1.4 days (p less than 0.0001). Since the survival in vivo is significantly less for platelets derived from the buffy coat stored at 4 degrees C, we advocate storage of platelets at 22 degrees C.     

The effect of the AcD-AG stabilizer on the survival time and surface properties of fluid preserved platelet concentrates compared to concentrates from fresh blood]

In the present investigations the storage effect the AcD-AG stabilizer on thrombocytes is examined. The thrombocytokinetic parameters of 9 fresh blood concentrates and 15 concentrates of AcD-AG plasma containing platelets were determined. Storage time amounted to three days. The results show that storage with AcD-AG is only possible to a limited degree. On an average the survival time of the platelets was reduced to 2.7 +/- 1.1 days compared with 9.0 +/- 1.0 days of fresh blood concentrates. The recovery of the stored platelets amounted to 25.3 +/- 16.1%, that of the fresh blood concentrates to 63.3 +/- 23.6%. The spleen-heart quotients and those of the liver-heart or the surplus impulses over the spleen and liver respectively indicate that there is a predominant destruction in the spleen for those thrombocytes stored for three days. The liver is scarcely involved in this sequestration process. With 36.1% platelet yield was very low in concentrates gained from AcD-AG plasma containing platelets and having been stored for 3 days. In cases of emergency a clinical application of concentrates prepared in this way should not be given up. If being used, the greater requirement has to be taken into account. If the substitution therapy is continued, however, fresh blood concentrates have to be used as soon as possible.     

Time course of the age-related alterations in stored blood

The extent and time course of the impairments occurring in whole blood and erythrocyte cells stored under blood bank conditions were studied by monitoring the reduction of MAL-6 spin label added to the media containing whole blood or erythrocyte cells using electron spin resonance (ESR) technique. Impairments forming in the erythrocyte cells incubated for various times at 37 degrees C were also studied. Erythrocyte cells were found to undergo changes during the storage or incubation, leading to fast decay of MAL-6 spin labels signal height. The extent of the changes depends on storage or incubation time. However, the reduction in incubated or artificially aged erythrocyte (AAE) cells was faster than the reduction in whole blood (WB) and aged erythrocyte (AE) cells stored under blood bank conditions. Two exponential curves attributed to the liquid and cellular parts of a given samples were found to be described best in the reduction of MAL-6 spin label in WB, AE and AAE.     

Leukocyte-depleted blood: a comparison of available preparations.

Febrile nonhemolytic transfusion reactions due to leukoagglutinins are frequently seen in patients who have been given multiple blood transfusions. To prevent or reduce the severity of these reactions, leukocyte-poor blood (that containing fewer than 0.3 X 10(9) leukocytes per unit) is frequently requested by clinicians. Four methods commonly used in Canada to produce leukocyte-poor blood were examined for their relative effectiveness and appropriate use. The mean total leukocyte count per unit was reduced to 0.22 X 10(9) in buffy-coat-poor red blood cell preparations produced by centrifugation with the blood bag inverted, to 0.19 X 10(9) by perfusion through an Imugard filter, to 0.21 X 10(9) by the use of an IBM 2991 automated cell washer and to 0.13 X 10(9) with the use of frozen blood. The proportion of red cells recovered varied from 62% with the inverted-spin method to 85% with the use of frozen blood. Comparison of these data and the percentage of leukocytes removed, the shelf life of the product, the cost of supplies and the preparation time indicated that the use of sophisticated machinery, such as the IBM cell washer, or of glycerolization plus washing of frozen cells is not warranted for most patients. Instead, patients who have febrile nonhemolytic transfusion reactions should initially be treated with a leukocyte-poor red cell preparation produced by the inverted-spin method; only if such reactions recur should the blood bank be requested to provide filtered, washed or frozen red cells.     

Incorporation of the whole chromosomal DNA in protoplast lysates into competent cells of Bacillus subtilis

Competent cells of Bacillus subtilis AC870 (purB, leuB, trpC, ald-1) were transformed to Ade+, Trp+, or Ade+ Trp+ with DNA in protoplast lysates of B. subtilis AC819 (hisH, tet-1, rpsL, smo-1). The cotransfer ratio of purB to trpC was constant at 7-9% (Ade+ Trp+/Trp+) or 3% (Ade+ Trp+/Ade+) at protoplast concentrations of 2.7 x 10(3) to approximately 2.7 x 10(6) per ml. The whole chromosomal DNA must be certainly incorporated into competent cells from the following reasons; (1) purB is opposite to trpC on the chromosome, (2) 2.7 x 10(3) protoplasts per ml is about 100 times lower than 3.2 x 10(5) competent cells per ml, and (3) the cotransfer ratio is constant at all the concentrations. Similar results were obtained with the cotransfer ratio of purA to trpC. The transformation requires several Com proteins including ComK.     

Collection of hematopoietic stem cells from canine peripheral blood and their ability to regenerate hematopoiesis]

A procedure is presented for the collection of a large number of hemopoietic stem cells from the peripheral blood of dogs by means of a single leukapheresis using the NCI-IBM Blood Cell Separator. In the course of a leukapheresis of about 285 min duration a mean of 23 x 10-9 leukocytes is collected from the blood. The hemopoietic stem cells among such separated leukocytes initiate repopulation of bone marrow within 10 days after whole body X-irradiation with 1200 R. The cell numbers in a defined histological section of femoral bone marrow are evaluated 9 to 10 days after irradiation and subsequent autologous transfusion of 6.72 x 10-9 separated mononuclear leukocytes. The results indicate that the bone marrow cell numbers of transfused dogs are significantly greater than in dogs given only 1200 R and reach a level of approximately 49% of the normal value. Possible ways of increasing the yield of hemopoietic stem cells from the peripheral blood will be considered.     

Interaction of liposomes with human leukocytes in whole blood

The uptake of multilamellar liposomes into human leukocytes in whole blood in vitro was evaluated on the basis of the cellular association of liposomal markers (3H-labelled cholesterol, lipid phase; [14C]inulin, aqueous phase). The entry of liposomes into human blood leukocytes was linear for 60 min and was mediated by a saturable mechanism displaying affinity constants of 0.28 +/- 0.17 and 0.16 +/- 0.05 mM liposomal lipid (means +/- S.E.) for liposomal lipid and aqueous phase markers, respectively. Amicon filtration analysis of incubation mixtures containing blood and liposomes (phosphatidylcholine:dicetyl phosphate:cholesterol, 70:20:10) showed that 34% of [14C]inulin was lost (neither liposome-associated nor cell-associated) after 60 min. By preincorporating sphingomyelin (35 mol%) into multilamellar liposomes, the leakage of the model aqueous phase marker inulin was reduced to 8% after 60 min, thus enhancing the drug carrier potential of liposomes in blood. As a consequence of their interaction with liposomes, the polymorphonuclear leukocytes in whole blood decreased in apparent buoyant density, while maintaining their viability. These results indicate that blood leukocytes in their natural milieu of whole blood are capable of interacting with, and taking up multilamellar liposomes.     

Leukocyte supplementation increases the luteinizing hormone-induced ovulation rate in the in vitro-perfused rat ovary

Ovaries from eCG-primed (20 IU s.c. on Day 28) rats were perfused from the morning of Day 30 of age in a recirculating system initially containing a buffered blood cell-free medium (M199 + 4% BSA) for periods of up to 21 h. The addition of ovine LH (0.1 micrograms/ml) at 0 h of perfusion resulted in ovulations in all 6 ovaries perfused (3.2 +/- 0.7 ovulations per treated ovary; mean +/- SEM), whereas none of the 6 control ovaries ovulated. Rat leukocytes (50 x 10(6)), added at 7 h of perfusion significantly increased the number of LH-induced ovulations (7.8 +/- 0.5; p less than 0.05). All ovulated oocytes showed resumption of meiosis as judged from the presence of germinal vesicle breakdown. Ovaries perfused with leukocytes but without LH did not ovulate. Histological examination of ovaries 14 h after leukocyte administration showed a considerable number of perifollicular extravasated white blood cells. These findings indicate that leukocytes participate in the normal ovulatory process as part of an inflammation-like reaction.     

Preparation of leukocyte-poor platelet concentrates from buffy coats by a new type of quadruple bag

A new quadruple blood bag system for collection of platelet concentrates is described. The principle modification consists of a smaller (150 ml) buffy coat bag in conjunction with SAG-mannitol for red cell preservation. Platelet yield and function were comparable to conventional techniques, leukocyte contamination was lower (1.7 +/- 1.3 x 10(7) per unit). The system is recommended for specialized institutions with a high demand of blood components.     

Preparation of leukocyte-poor erythrocyte concentrates using a simple aspiration technic

Depletion of leukocytes from red blood cells can prevent transfusion reactions in HLA-sensitized patients. We describe a simple technique of aspiration of the buffy-coat after centrifugation of red blood cells concentrates, less than 6 days old. This method can remove 87% of the leukocytes. The average leukocyte count after aspiration is 2 x 10(8). This method is inexpensive and does not require any special equipment.     

Pattern and rate of indel evolution inferred from whole chloroplast intergenic regions in sugarcane, maize and rice.

Microstructural changes such as insertions and deletions (=indels) are a major driving force in the evolution of non-coding DNA sequences. To better understand the mechanisms by which indel mutations arise, as well as the molecular evolution of non-coding regions, the number and pattern of indels and nucleotide substitutions were compared in the whole chloroplast genomes. Comparisons were made for a total of over 38 kb non-coding DNA sequences from 126 intergenic regions in two data sets representing species with different divergence times: sugarcane and maize and Oryza sativa var. indica and japonica. The main findings of this study are: (i) Approximately half of all indels are single nucleotide indels. This observation agrees with previous studies in various organisms. (ii) The distribution and number of indels was different between two data sets, and different patterns were observed for tandem repeat and non-repeat indels. (iii) Distribution pattern of tandem repeat indels showed statistically significant bias towards A/T-rich. (iv) The rate of indel mutation was estimated to be approximately 0.8 +/- 0.04 x 10(-9) per site per year, which was similar to previous estimates in other organisms. (v) The frequencies of nucleotide substitutions and indels were significantly lower in inverted repeat (IR).     

Leukocyte counting from a small amount of whole blood using a size-controlled microcavity array

Absolute counting of total leukocytes or specific subsets within small amounts of whole blood is difficult due to a lack of techniques that enable separation of all leukocytes from limited amounts of whole blood. In this study, a microfluidic device equipped with a size-controlled microcavity array for highly efficient separation of leukocytes from submicroliters of whole blood was developed. The microcavity array can separate leukocytes from whole blood based on differences in the size and deformability between leukocytes and other blood cells. Leukocytes recovered on aligned microcavities were continuously processed for image-based immunophenotypic analysis. Our device successfully recovered over 90% of leukocytes in 1 μL of whole blood without pretreatment such as density gradient centrifugation or erythrocyte lysis. In addition, the proposed system successfully performed absolute enumeration of human CD4(+) and CD8(+) leukocytes from 1 μL of whole blood, and the obtained data showed good correlation with conventional flow cytometric analysis. Our microfluidic device has great potential as a tool for a point-of-care leukocyte analysis system.     

Alterations by leukocytes of erythrocyte flow in microchannels

The effects of leukocytes on blood rheology in the microcirculation may be predicted to result from the rheological characteristics of the individual white blood cell types and their behavior at entrances of small vessels; their influence on flow resistance of blood due to their disturbance of erythrocyte flow; and the obstruction caused by their adherence to endothelial cells or geometrical hindrance of their flow at irregular or narrow regions. Deformation of leukocytes in micropipettes indicates the relative rigidity of lymphocytes and blast cells from leukemias in contrast to granulocyte viscoelastic properties, and entry times for lymphocytes in 2.6-4.6-microns channels were 11-151 s but milliseconds in 9.1-micron tubes. Lymphocytes and erythrocytes rarely (less than 1%) adhered to cultured endothelium; however, granulocytes adhered avidly at shear stresses of 10-100 dyn/cm2, typical of microcirculation. In the 9.1-microns flow tube at Hct = 17.7 +/- 2.6%, increasing [WBC] caused a linear increase in flow resistance, but above [WBC] = 1000/mm3 resistance tended to plateau. These data support the interpretation that granulocyte adherence to endothelial cells with the potential for obstruction of flow may be a more significant rheological consequence of leukocytes in blood than their resistance to flow because of their relative rigidity or their influence on flow resistance by perturbation of the erythrocyte flow.     

Storage-induced changes in erythrocyte membrane proteins promote recognition by autoantibodies

Physiological erythrocyte removal is associated with a selective increase in expression of neoantigens on erythrocytes and their vesicles, and subsequent autologous antibody binding and phagocytosis. Chronic erythrocyte transfusion often leads to immunization and the formation of alloantibodies and autoantibodies. We investigated whether erythrocyte storage leads to the increased expression of non-physiological antigens. Immunoprecipitations were performed with erythrocytes and vesicles from blood bank erythrocyte concentrates of increasing storage periods, using patient plasma containing erythrocyte autoantibodies. Immunoprecipitate composition was identified using proteomics. Patient plasma antibody binding increased with erythrocyte storage time, while the opposite was observed for healthy volunteer plasma, showing that pathology-associated antigenicity changes during erythrocyte storage. Several membrane proteins were identified as candidate antigens. The protein complexes that were precipitated by the patient antibodies in erythrocytes were different from the ones in the vesicles formed during erythrocyte storage, indicating that the storage-associated vesicles have a different immunization potential. Soluble immune mediators including complement factors were present in the patient plasma immunoprecipitates, but not in the allogeneic control immunoprecipitates. The results support the theory that disturbed erythrocyte aging during storage of erythrocyte concentrates contributes to transfusion-induced alloantibody and autoantibody formation.     

Exercise-induced hemolysis is caused by protein modification and most evident during the early phase of an ultraendurance race.

Whether structural changes of the erythrocyte membrane increase the susceptibility to hemolysis particularly of the relatively older cell population during the early phase of a 216-km ultrarace was tested in six male runners (age 53.6 +/- 10.4 yr, height 175.8 +/- 11.1 cm, body mass 75.9 +/- 8.4 kg). Erythrocyte membrane spectrins were lowest (P < 0.001) after 42 km (75.59 +/- 5.25% of prerace) and increased (P < 0.001) toward 216 km (88.27 +/- 3.37%). Susceptibility to osmotic hemolysis was highest (P < 0.01) after 42 km (107.34 +/- 3.02 mOsm sodium phosphate buffer) with almost identical (P > 0.05) values prerace (97.98 +/- 3.41 mOsm) and postrace (98.61 +/- 3.26 mOsm). Haptoglobin indicated intravascular hemolysis of 9.27 x 10(9) cells/l (P < 0.05) during the initial 84 km. Changes in hematocrit and plasma proteins indicated an estimated total net erythrocyte loss of 3.47 x 10(11) cells/l (P < 0.05) after 21 km. This was compensated by a gain in erythrocytes (P < 0.05) of 3.31 x 10(11) cells/l during the final 132 km. A main effect (P < 0.05) on erythropoietin suggests increased erythropoiesis throughout the race. Exercise-induced hemolysis reflects alterations in erythrocyte membrane spectrins and occurs particularly in the early phase of an ultraendurance race because of a relative older cell population.     

The effect of diet on ouabain binding to erythrocytes from obese subjects

Ouabain binding to erythrocyte membranes is increased in obese subjects. Three study groups are compared: 14 reference subjects, 102 +/- 16% of ideal weight; 9 obese on unrestricted diets, 207 +/- 16% of ideal weight; 11 obese on restricted diets, 202 +/- 35% of ideal weight. A reproducible (CV = 11.3%) ouabain-binding assay is used to measure Na+-K+ ATPase sites in erythrocyte membranes. The number of binding sites per red blood cell for obese subjects on unrestricted diets, 431 +/- 30, is greater than for the reference group, 346 +/- 66 (p less than 0.01), or for obese subjects on restricted diets, 371 +/- 68 (p less than 0.05). These data suggest that caloric intake influences the number of Na+-K+ ATPase sites. Scatchard plots indicate only one type of binding site for ouabain with an affinity constant of about 3 X 10(8) M-1.     

State and development of liquid conservation of thrombocytes]

In the present investigation the storage effect of AcD-AG and CPD-AG-stabilizers on thrombocytes was tested. The platelets were stored in platelet-rich plasma (PRP) at 4 degrees C or room temperature for 3 days. The concentrates gained by it were marked with Na251CrO4 and reinjected. The thrombocytokinetic parameters were evaluated. The results show that storage with the help of the mentioned stabilizers can be made to a certain extent only. Platelets stored in AcD-AG stabilizerhad a survival time of 2.7 +/- 1.1 days towards 9.0 +/- 1.0 days of fresh whole blood concentrates. The survival time of CPD-AG thrombocytes stored at 4 degrees C for 3 days amounted to 2.0 +/- 0.5 days. Storage of CPD-AG platelets at room temperature showed favourable results. Their survival time amounted to 6.2 +/- 0.6 days. Measurements of surface activity above the spleen and the liver indicate that degradation of stored platelets is mainly performed in the spleen. Problems of liquids storing in view of the significance of therapeutic thrombocyte substitution for hospitals are referred to.