Collection, storage and transfusion of blood stem cells for the treatment of hemopoietic failure.

Migration of hemopoietic stem cells via the blood to sites of stem cell need is a principle that becomes established during the embryonic development of hemopoiesis and can be observed in the adult whenever bone marrow transplantations are being performed. The regular presence of stem cells in the peripheral blood lends itself to the study of their collection, storage, and use for transfusion purposes in cases of bone marrow failure. Both in dog and in man, granulocyte-macrophage progenitor cells (CFU-C) can be collected by leukapheresis from the blood in large quantities, particularly if the yield is increased by the administration of mobilizing agents such as dextran sulfate, and appear to be an indicator for the presence of stem cells. For collection and storage, a closed plastic bag system has been developed that allows the safe handling of the cells. The loss of CFU-C from freezing and thawing with DMSO as a cryoprotective agent is only 10%-20%. If frozen and thawed mononuclear leukocytes are transfused into 1200 rad whole-body X-irradiated autologous or allogeneic recipient dogs, a hemopoietic take is observed when 0.2 X 10(5) CFU-C are present among the mononuclear leukocytes (MNC). Graft-versus-host disease can be avoided in the allogeneic situation when a purified CFU-C rich cell fraction is being transfused. In man collection and storage of MNC including CFU-C is feasible and may eventually become a therapeutic tool.     

Preservation of erythrocytes by freezing in liquid nitrogen. Use of an I.B.M. blood regenerator]

The freezing of blood permits preservation of red cells over long periods of time, several months or years. Leucocyte and platelet contamination of red cell concentrates to be frozen is negligible. The amount of the various red cell metabolites (2.3 D.P.G., A.T.P., etc.) is maintained. Washing of thawed red cells removes the remaining plasma proteins and cell residues. The freezing method employed is that of Row et al. The protector used is 28% glycerol added in equal amounts to red cell concentrate to be frozen. The blood bag is kept in liquid nitrogen at -- 196 degrees C. Thawing takes place in a water bath at 45 degrees C. Wash solution is the IBM Blood regenerator. The solution used for removing glycerol is hypertonic natrium chloride. The following parameters have been investigated: --hemoglobin level; --osmotic fragility; --the amount of 2.3 D.P.G.; --residual glycerol after thawing; and clearance of leucocytes and platelets following each step of the protocol. Preliminary data regarding these features and therapeutic efficiency of processed blood are satisfactory.     

Recovery,structure, and function of dog granulocytes after freeze-preservation with dimethylsulfoxide

The recovery, structure and function of dog granulocytes were determined before and after freeze-preservation. Leucocytes were isolated from defibrinated or anti-coagulated whole blood and subsequent erythrocyte sedimentation on a column of 2:1 dextran (6%)-isopaque (33.9%). Granulocytes isolated by these procedures were examined for changes in O₂ consumption associated with phagocytosis, in vitro directed migration (chemotaxis), bactericidal activity, and ultrastructure before and after freezing. Granulocytes were frozen in DMSO (7.5%) and autologous serum or HBSS-minus and 20% autologous serum at the rate of ?1 °C/min to ?80 °C and stored in liquid N₂ vapor.After freeze-preservation, O₂ consumption associated with phagocytosis was decreased by 54 and 64% for granulocytes isolated from defibrinated or from ACD-anticoagulated blood, respectively. Bactericidal activity is only slightly depressed in samples from either isolation method after freeze-preservation when compared to the prefreeze controls, but granulocytes isolated from defibrinated blood are significantly less effective in killing bacteria than those from ACD-anticoagulated blood. Chemotactic response after freeze-preservation was completely inhibited in granulocytes isolated from defibrinated blood. Exposure of granulocytes to ACD inhibited chemotaxis prior to freezing, but the granulocytes responded chemotactically after freeze-thaw and additional washing. The ultrastructure of granulocytes observed before and after freeze-thaw was similar for cells isolated by both methods. However, nuclear, cytoplasmic, and granular changes observed were slightly greater in granulocytes isolated from defibrinated blood. Dog granulocytes isolated by either method withstood freeze-preservation in DMSO to a degree not previously reported.It is concluded that dog granulocytes freeze-preserved by these methods are functional in vitro, but that phagocytic, directed migration, and bactericidal functions and ultrastructure are impaired to different degrees, according to the method of isolation and preparation for storage. These results indicate the need for continued investigation on the effects of storage variables on the preservation of granulocytes.     

Comparison of chemical dehydration and critical point drying for the stabilization and visualization of aging biofilm present on interior surfaces of PVC distribution pipe

In this study, fixation of attached glycocalyx on the interior surfaces of polyvinyl chloride distribution pipe remnants was compared with and without ruthenium red/osmium tetroxide and, in the final preparatory phase, with chemical dehydration and critical point drying. SEM examination of interior surface of the polyvinyl chloride pipe showed varying concentrations of adherent bacteria, depending on the preparatory technique used. It was concluded that using a combination of ruthenium red/osmium tetroxide and critical point drying is the optimum method for visually demonstrating aging biofilm on the interior surface of contaminated polyvinyl chloride pipe.     

Fructose formation in stored blood

At the high glucose concentrations used in the collection and storage of donor blood the activity of the fructose-forming polyol pathway (Reaction I and II) could act to deplete NADPH and thus GSH, thereby exposing the cells to oxidative stress. Fructose levels were found to be high in red cells and the supernatant plasma of blood collected into CP2D, which contains 258 mM glucose. Elevated fructose was not produced by the polyol pathway, but was formed by the autoclaving process. A high fructose concentration sufficient to account for the fructose in donor red cells was also found in the CP2D anticoagulant and in samples of autoclaved glucose.     

Response of cytoskeleton of murine osteoblast cultures to two-step freezing

Understanding the ultrastructural response of cells to the freezing process is important for designing cryopreservation strategies for cells and tissues. The cellular structures of attached cells are targets of cryopreservation-induced damage. Specific fluorescence staining was used to assess the status of the actin filaments （F-actin） of murine osteoblasts attached to hydroxyapatite discs and plastic coverslips for a two-step freezing process. The F-actin of dead cells was depolymerized and distorted in the freezing process, whereas that of live cells had little change. The results suggest that the cytoskeleton may support the robustness of cells during cryopreservation. The present study helps to investigate the damage mechanism of attached cells during the freezing process.     

A novel device for the non-invasive measurement of free hemoglobin in blood bags.

The production of red blood cell concentrates from human donors is a very expensive procedure and human resources are in short supply. Under perfect storage conditions at a temperature of 2-6 degrees C, a blood bag must be used within 35-49 days (in Germany). Visual inspection of the bag for apparent hemolysis by a blood bank physician is a crucial but subjective quality control assessment. Since an interruption of the cold chain cannot be definitely ruled out, bags are often disposed of prematurely for safety reasons. There is currently no method of testing a closed blood bag with respect to hemolysis for its suitability to be used in a transfusion. The proposed optical measuring device is a hemoglobin sensor which determines the free hemoglobin in standard erythrocyte concentrates without opening the bag. The optical measurements are done on the flexible tube connected to the main bag. The optical measurements were evaluated using standard hemoglobin solutions with an accuracy of 0.005 g/dL. These investigations show that in the future each blood bag can be tested non-invasively for its content of free hemoglobin. This will contribute to decreasing the wastage rate of red blood cell concentrates.     

Diethylhexyl phthalate in plastic bags and glass bottles for blood preservation]

The amount of di-(-2 ethyl hexyl) phtalate (D.E.H.P.) is measured in the liquid contained in plastic bags or glass bottles for blood storage. Comparisons between the results show that D.E.H,P. has an equal level in plastic and glass container liquid except for one blood packs series. Particular attention has been paid to obtain solvents and glassware free from D.E.H.P., and to the specificity and the sensitivity of the detection method: a mass fragmentographic technic has been used.     

Biochemical parameters to assess viability of blood stored for transfusional use

Blood collected in citrate-phosphate-dextrose-adenine (CPDA-1) containing transfusional bags, was weekly tested throughout a 35 day period. Biochemical assays included plasmatic glucose, electrolytes, free Hb, acid-base balance and hemogasanalysis. Intraerythrocytic ATP and 2,3 DPG were also determined. Results show that an almost total 2,3 DPG depletion occurs during the first three weeks, whereas intracellular ATP are about 50% of the initial values, at the same time. Lowering of pH is also maximal at the third week. pCO2 variation pattern is biphasic: an early increase due to HCO3- titration by lactic acid arising from red cells glycolysis, followed by a decrease probably due to plastic bag permeability to CO2 itself. The percentage of O2Hb also rises during blood storage: this might be the combined result of increase in pO2 and decrease in 2,3 DPG content. A rise of free Hb was obtained; extracellular K+ levels underwent a sixfold increase in 35 days. The mechanism of relative variations of these parameters and the gas transport are discussed. Some of these parameters might be used as routine controls to asses viability and functional status of stored red cells for transfusional use.     

Hemolysis of human red blood cells by freezing and thawing in solutions containing polyvinylpyrrolidone: relationship with postthypertonic hemolysis and solute movements

An investigation was made into the effects of the presence of polyvinylpyrrolidone (PVP) on changes in human red blood cells suspended in hypertonic solutions, on posthypertonic hemolysis, and on freezing at temperatures down to ?12 °C.PVP is very effective at reducing hemolysis when the red blood cells are frozen at temperatures down to ?12 °C. However, the membranes of the cells recovered on thawing have become very permeable to sodium and potassium ions and there is a much increased hemolysis if the cells are resuspended in an isotonic solution of sodium chloride.The presence of PVP does not affect the dehydration of the cells or the development of a change in membrane permeability when the cells are shrunken in hypertonic solutions at 0 °C. Neither does its presence in the hypertonic solution reduce the extent of posthypertonic hemolysis at 4 °C (as measured by the hemolysis on resuspension in an isotonic solution of sodium chloride), but it is more effective than sucrose at reducing hemolysis when present in the resuspension solution. It is concluded that the PVP is able to prevent swelling and hemolysis of cells which are very permeable to cations by opposing the colloid osmotic pressure due to the hemoglobin. However, this does not explain how PVP is able to protect cells against freezing damage at high cooling rates, and a mechanism by which it might do this is discussed.     

Effect of blood admixture on in vitro survival of chilled and frozen-thawed canine spermatozoa

Hematospermia in the dog usually occurs secondary to benign prostatic hypertrophy or trauma of the penis or prepuce during semen collection. Regarding the difficulty of removing blood cells from a hematospermic sample, the present study was performed to determine whether blood contaminated ejaculates can still be chilled (4 degrees C) or frozen (-196 degrees C) without an additional decrease in sperm quality. In the first experiment, blood additions of up to 10% exerted no negative effects on the functional characteristics of canine spermatozoa cooled (4 degrees C) and stored for 4 days in an egg-yolk-Tris extender. In contrast, in experiment 2, blood admixtures of 4% or more clearly caused negative effects on cryopreserved (-196 degrees C) spermatozoa, mainly on the motility parameters, on the membrane integrity and on the acrosomal status of the spermatozoa. In experiment 3, we showed that these negative effects of blood admixture on cryopreserved spermatozoa were mainly associated with the red blood cells (RBCs) whereas the addition of plasma, serum or inactivated serum exerted little or no negative effect. Moreover, in experiment 4, we showed that 58.3+/-11.6% of the RBCs hemolysed after a freeze-thaw process. In experiment 5, a clear and negative effect of hemoglobin on cryopreserved canine spermatozoa was observed. We conclude that the presence of up to 10% blood is not detrimental for the storage of chilled canine spermatozoa and that the detrimental effects of blood on cryopreserved spermatozoa are at least partly attributable to the high amount of hemoglobin originating from the RBC hemolysis observed after freezing and thawing.     

Bioequivalence comparison of a new freezing bag (CryoMACS(®)) with the Cryocyte(®) freezing bag for cryogenic storage of human hematopoietic progenitor cells

Background aimsWe investigated two different plastic freezing bags, namely the most recently U.S. Food and Drug Administration (FDA)-approved CryoMACS^® freezing bag (200-074-402) from Miltenyi Biotec and the familiar Cryocyte^® freezing bag (R4R9955) from (Baxter Healthcare, Deerfield, IL, United States) for the cryogenic storage of human hematopoietic progenitor cells (HPC).MethodsThe study material consisted of 12 frozen HPC pairs (= 24 transplant units) that were no longer needed for autologous treatment of patients. After thawing, one unit of a pair was transferred into the Miltenyi (M) bag; the other unit remained in the original Baxter (B) bag. After refreezing both units, all units were stored again under cryogenic conditions either partially immersed in liquid nitrogen (n = 22) or in the vapor phase over liquid nitrogen, n = 2, ResultsThe correlation coefficients (r) between the results obtained from the two bag types were high for white blood cells (WBC) content (r = 0.98), mononuclear cells (MNC) (r = 0.97), lymphocytes (r = 0.98), monocytes (r = 0.96), membrane integrity (r = 0.93), concentration of ‘free’ hemoglobin (r = 0.97) and hemolysis rate (r = 0.95). With regard to clonogenicity, there were no significant differences (Student's paired t-test) for the three parameters investigated [i.e. total number of colonies, including the numbers of burst-forming units–erythroid (BFU-E) and colony-forming units–granulocyte-macrophage (CFU-GM) colonies, respectively).ConclusionsThe CryoMACS freezing bag 200-074-402 is bioequivalent to the Cryocyte freezing container R4R9955. An advantageous feature of the CryoMACS is that its double-sterile wrapping provides additional safety regarding potential cross-contamination during cryogenic storage.     

Immunocompetence of the residual lymphocytes in frozen blood

Lymphocytes remaining in washed, previously frozen blood were isolated and their immunocompetence was studied on the basis of T- and B-cell markers, mitogenic responses, and mixed lymphocyte culture (MLC). The results showed that freeze-preservation of red cells considerably deteriorated immunocompetence of the residual lymphocytes in addition to preferential removal of polymorphonuclear cells. While approximately 60% of the residual lymphocytes excluded trypan blue dye, their E and EAC rosette formation decreased markedly to 6.4 and 5.2%, respectively.These lymphocytes showed weak proliferation to PHA and Con A stimulation, but failed to respond to alloantigens in MLC. In contrast, they retained stimulating activity to allogeneic lymphocytes in MLC. These observations suggest that some portion of the residual lymphocytes may remain viable enough to possess immunocompetence and recognizable immunogenicity.     

The "unfrozen fraction" hypothesis of freezing injury to human erythrocytes: a critical examination of the evidence

This paper examines the experimental evidence presented by Mazur and his colleagues to support their hypothesis that the survival of slowly frozen human red blood cells is primarily dependent on the fraction of water that remains unfrozen, rather than on the high concentrations of sodium chloride produced by the formation of ice. This hypothesis is in direct conflict with the general belief that freezing injury under such conditions is caused by the concentration of solutes in the solution surrounding the cells: if the "unfrozen fraction" hypothesis is true, then much of the evidence supporting that belief must be dismissed as mere coincidence. We have reexamined Mazur's data, and have suggested an alternative explanation--that cells which are initially suspended in solutions that are not isotonic differ in their susceptibility to subsequent freezing and thawing, shrunken cells being more resistant and swollen cells more susceptible than normal cells. If this is true then the data can be explained without invoking a direct effect of the unfrozen fraction, solely on the basis of changes in the concentration of the solution surrounding the cells. We cite other experimental evidence, obtained in the absence of freezing, that red blood cells do indeed possess the required property. We further argue that the known effects of variations in cooling and warming rate, and in hematocrit, are able to account for the features observed by Mazur and his colleagues in their three published studies.     

Release of diethyl-2-hexyl phthalate from stored blood on contact with polyvinyl chloride

Softness and flexibility of PVC are due to the addition of plasticizers in high concentration; the most used of them for blood storage bags is DEHP. In this work, a method for labelling DEHP with 14C from 14C phtalic anhydrid is given. A piece of PVC from a commercial blood bag is labelled, in our laboratory, with 14C DEHP and used to follow the kinetics of DEHP leaching in blood during storage. It is also used to study the influence of some parameters such as lipids amount of blood, shaking, and plastic sterilisation on this leaching. DEHP leaching is a three steps phenomenon and the level is not correlated to lipids content of blood. Thermal treatment of PVC and shaking have an influence on leaching. DEHP is not metabolised during blood storage at 4 degrees C and can not be detected as free molecule; it is absorbed on plasmatic proteins.     

Kinetic characteristics of the sulfate self-exchange in human red blood cells and red blood cell ghosts

Summary The sulfate and the chloride self-exchange fluxes were determined by measuring the rate of the tracer efflux from radioactively labeled human red blood cells and red blood cell ghosts. The concentration dependence and the pH-dependence of the sulfate self-exchange flux were studied. In addition, the effects of some monovalent and divalent anions on the sulfate and the chloride self-exchange fluxes were investigated.The sulfate self-exchange fluxes saturate, exhibiting a concentration maximum at sulfate concentrations between 100 and 300mm (25°C). The position of the concentration maximum depends upon pH. At high sulfate concentrations a self-inhibition of the flux becomes apparent. The apparent half-saturation constant and the apparent self-inhibition constant at pH 7.2 were 30mm and 400mm respectively. Within the pH range of 6.3–8.5, both constants decreased with increasing pH. No saturation of the sulfate self-exchange flux was observed if the sulfate concentration was raised by substituting sulfate for isoosmotic amounts of a second salt (NaCl, NaNO₃, Na-acetate, Na-lactate, Na-succinate or Na₂HPO₄). Red blood cells and red blood cell ghosts display the same pattern of concentration responsiveness.The sulfate self-exchange flux exhibits a pH-maximum at about pH 6.2 (37°C). The location of the pH-maximum is little affected by variations of the sulfate concentration. The logarithmic plots (log vs. pH) revealed that the flux/pH relation can be approximated by two straight lines. The slopes of the alkaline branches of the flux/pH curves range from –0.55 to –0.86, the slopes of the branches of the curves range from 0.08 to 1.14 and were strongly affected by changes of the sulfate concentrations. The apparent pK's obtained from the alkaline and from the acidic branches of the flux/pH curves were about 7.0 and 6.0, respectively. Intact red blood cells and red blood cell ghosts display the same type of pH-dependency of the sulfate self-exchange flux.The sulfate self-exchange flux is competitively inhibited by nitrate, chloride, acetate, oxalate and phosphate. The chloride self-exchange flux is competitively inhibited by thiocyanate, nitrate, sulfate and phosphate. The inhibition constants for the various anion species increase in the given sequence.The results of our studies indicate that the sulfate self-exchange flux is mediated by a two-site transport mechanism consisting either of a mobile carrier or a two-site pore. The experiments reported in this paper do not permit distinguishing between both transport mechanisms. The similarities of the sulfate and the chloride self-exchange flux and the mutual competition between sulfate and chloride point to a common transport system for both anion species.     

Droplet freezing of antibody-linked indicator red cells of sheep, ox, and human origin

A droplet freezing technique for the cryopreservation of indicator red cells is described. Recovery was crucially dependent on the composition of the solution in which the cells were suspended. Preliminary experiments to determine the relative importance of sucrose, glucose, sodium chloride and hydroxyethyl starch (HES) in determining the survival of trypsin-treated sheep red cells showed that the addition of sucrose or HES or both to isotonic sodium chloride solution increased recovery, whereas the additional inclusion of glucose was detrimental. It was shown that glucose penetrated the cells whereas sucrose did not. The optimum combination of sucrose and sodium chloride concentration, in the presence of 6 g/dl HES, was 7 g/dl sucrose plus 0.3 g/dl sodium chloride. Recovery was increased by increasing the concentration of HES, and maximal recovery was obtained by thawing the frozen droplets in phosphate-buffered saline at 40 °C. Trypsintreated ox and human cells gave much lower recovery than sheep cells when HES was used in the freezing mixture but the substitution of dextran (10 g/dl) for HES gave greater than 80% recovery with all three species. Ten different antibody-coupled reagent cells all gave >83% recovery. The effects of hematocrit, incubation time, and storage temperature are described. The preservation technique described is simple and convenient, and will make it possible to extend the use of immunoassay procedures using antibody-coupled red cells.     

Clinical use of blood,blood components and blood products.

The goal of modern transfusion therapy is to provide appropriate replacement therapy with blood components as opposed to whole blood for patients with specific hematologic deficiencies. A prerequisite of component therapy is, therefore, correct identification of the deficiency. Appropriate use of components avoids many of the hazards associated with the use of whole blood, and at the same time makes maximal use of this valuable resource. Blood components separated from whole blood soon after collection and appropriately stored can, in combination, provide all the factors present in fresh whole blood. Red cell concentrates prepared from multiple packs have a hematocrit of approximately 70%. They may be stored for up to 3 weeks at 4 degrees C and are recommended for most situations requiring red cell transfusions. Platelet concentrates, which can be stored for up to 72 hours at 22 degrees C, may be used for thrombocytopenic patients. Fresh frozen plasma, stored plasma, cryoprecipitated factor VIII, factor VIII concentrate and factor IX complex concentrate are available for the proper treatment of patients with hemorrhagic disorders due to coagulation factor deficiencies. Similarly, albumin and immune serum globulin are available for their oncotic and antibody properties respectively. Thus, the availability and appropriate use of the various blood products allows not only optimal transfusion therapy for each patient, but also fuller utilization of national blood resources.     

Leucocytes isolated from simply frozen whole blood can be used in human biomonitoring for DNA damage measurement with the comet assay

Preservation of human blood cells for DNA damage analysis with the comet assay conventionally involves the isolation of mononuclear cells by centrifugation, suspension in freezing medium and slow freezing to ?80 °C—a laborious process. A recent publication (Al‐Salmani et al. Free Rad Biol Med 2011; 51: 719–725) describes a simple method in which small volumes of whole blood are frozen to ?20 or ?80 °C; on subsequent thawing, the comet assay is performed, with no indication of elevated DNA strand breakage resulting from the rapid freezing. However, leucocytes in whole blood (whether fresh or frozen) are abnormally resistant to damage by H₂O₂, and so a common test of antioxidant status (resistance to strand breakage by H₂O₂) cannot be used. We have refined this method by separating the leucocytes from the thawed blood; we find that, after three washes, the cells respond normally to H₂O₂. In addition, we have measured specific endogenous base damage (oxidized purines) in the isolated leucocytes, using the enzyme formamidopyrimidine DNA glycosylase. In a study of blood samples from 10 subjects, H₂O₂ sensitivity and endogenous damage—both reflecting the antioxidant status of the cells—correlated significantly. This modified approach to sample collection and storage is particularly applicable when the available volume of blood is limited and has great potential in biomonitoring and ecogenotoxicology studies where samples are obtained in the field or at sites remote from the testing laboratory. Copyright © 2013 John Wiley & Sons, Ltd.     

A new type of collapsible insect-surveillance light trap for sampling Diptera

The newly designed Army Collapsible Insect Surveillance trap for nocturnal collection of flying Diptera that are attracted by light is described. A polyvinyl chloride construction increases durability, while the capability to collapse from a height of 92.1 cm to 24.1 cm facilitates transportation and storage.