Measuring micro-interactions between coagulating red blood cells using optical tweezers

Agents that alter the dynamics of hemostasis form an important part in management of conditions such as atherosclerosis, cerebrovascular disease, and bleeding diatheses. In this study, we explored the effects of heparin and tranexamic acid on the efficiency of blood coagulation. Using optical tweezers, we evaluated the pN-range micro-interaction between coagulating red blood cells (RBCs) by measuring the minimum power required to trap them. By observing the mobility of RBCs and the intensity of cellular interactions, we found that the coagulation process can be separated into three phases. The effects of heparin and tranexamic acid were examined by observing variations in cellular interaction during the coagulation phases. Heparin attenuated the interaction between RBCs and prolonged the first phase whereas the samples containing tranexamic acid bypassed the first two phases and immediately proceeded to the final one.     

Normal saline-induced deoxygenation of red blood cells probed by optical tweezers combined with the micro-Raman technique

The use of normal saline for washing red blood cells and treating critically ill patients is a regular medical practice in hospital settings. An optical tweezer in combination with Raman spectroscopy is an analytical tool employed for the investigation of single cell dynamics, thus providing molecular fingerprint of the cell by optically trapping the cell at a laser focus. In this study, the impact of normal saline on individual human red blood cell was compared with that of blood plasma using Raman tweezers spectroscopy. Major spectral variations in the marker frequencies at 1209 cm⁻¹, 1222 cm⁻¹, 1544 cm⁻¹, and 1561 cm⁻¹ of the Raman spectrum of the treated cells imply that the transition of hemoglobin to the deoxygenated state occurs when 0.9% normal saline is used. This may result in serious implications in blood transfusion. The results obtained from the principal component analysis also displayed clear differentiation among the red blood cells diluted in normal saline and those diluted in plasma. In future studies, efforts will be made to correlate the deoxygenation status of red blood cells with various human disorders.

Micro-Raman spectroscopy of optically trapped live red blood cell demonstrates normal saline induced deoxygenation.     

Quantifying the influences of radiation therapy on deformability of human red blood cells by dual-beam optical tweezers

Radiation therapy is widely used as a treatment tool for malignancies. However, radiation-related complications are still unavoidable risks for off-target cells. Little is known about radiation therapy''s possible effects on mechanical features of the off-target cells such as human red blood cells (RBCs). RBCs are nucleus-free circulating cells that can deform without losing functionality in healthy conditions. Thus, to evaluate in vitro effects of radiation therapy on the healthy plasma membrane of cells, RBCs were selected as a primary test model. RBCs were exposed to clinically prescribed radiotherapy doses of 2 Gy, 12 Gy and, 25 Gy, and each radiotherapy dose group was compared to a non-irradiated group. Cells were characterized by stretching using dual-beam optical tweezers and compared using the resulting deformability index. The group receiving the highest radiation dose was found statistically distinguishable from the control group (DI_0Gy = 0.33 ± 0.08), and revealed the highest deformability index (DI_25Gy = 0.38 ± 0.11, p = 0.0068), while no significant differences were found for 2 Gy (DI_2Gy = 0.33 ± 0.08, p = 0.9) and 12 Gy (DI_12Gy = 0.31 ± 0.09, p = 0.2) dose groups. Based on these findings, we conclude that radiotherapy exposure may alter the deformability of red blood cells depending on the dose amount, and measurement of deformability index by dual-beam optical tweezers can serve as a sensitive biomarker to probe responses of cells to the radiotherapy.

Little is known about radiation therapy''s possible effects on mechanical features of off-target cells such as human red blood cells. Here, irradiated human red blood cells were stretched using dual-beam optical tweezers and compared using the resulting deformability index.     

Stretching of red blood cells using an electro-optics trap

The stretching stiffness of Red Blood Cells (RBCs) was investigated using a combination of an AC dielectrophoretic apparatus and a single-beam optical tweezer. The experiments were performed at 10 MHz, a frequency high enough to avoid conductivity losses, but below the second turnover point between positive and negative dielectrophoresis. By measuring the geometrical parameters of single healthy human RBCs as a function of the applied voltage, the elastic modulus of RBCs was determined (µ = 1.80 ± 0.5 µN/m) and compared with similar values of the literature got by other techniques. The method is expected to be an easy-to-use, alternative tool to determine the mechano-elastic properties of living cells, and, on this basis, to distinguish healthy and diseased cellsOCIS codes: (350.4855) Optical tweezers or optical manipulation, (230.2090) Electro-optical devices, (170.3880) Medical and biological imaging     

Preparation of leukocyte-poor red blood cells using the IBM 2991 blood cell processor

Leukocyte-poor red blood cells (RBCs) were prepared from one-to-seven-day-old whole blood using the IBM 2991 Blood Cell Processor. Blood was centrifuged at 3,000 rpm for 5 minutes on the initial processing cycle and for 1.5 minutes each subsequent cycle. The red blood cell override (RCO) control was used to remove portions of the leukocyte-rich red blood cell layer at the cell/liquid interface after separation. RBC loss was directly proportional to RCO time and averaged 5.4, 10.6, 15.6, 21.2, and 26.7 ml with RCO settings of 1.5, 2.5, 3.5, 4.5, and 5.5 seconds, respectively. A single RCO cycle removed 54.7, 66.0, 70.7, and 69.7 per cent of total leukocytes at RCO settings of 2.5, 3.5, 4.5, and 5.5 seconds, respectively. With double cycle processing at the same RCO times, 78.6, 88.9, 90.3, and 90.7 per cent of white blood cells were removed with loss of 12.4, 16.7, 22.4, and 25.4 per cent of RBCs respectively. Four-cycle processing with an initial 2.5-to-5.5-second RCO followed by three 1.5-second RCO cycles removed the greatest number of leukocytes (86.9, 93.3, 93.3, and 91.7 per cent, respectively) with corresponding RBC losses of 11.9, 17.2, 20.2, and 23.1 per cent. Total leukocytes contaminating the processed RBCs averaged 0.37, 0.18, 0.18, and 0.22 × 10⁹, respectively. The processing cycle with the 3.5-second initial RCO provided maximal leukocyte removal and minimal erythrocyte loss.     

Massive fetomaternal hemorrhage: clearance of fetal red blood cells after intravenous anti-D prophylaxis monitored by flow cytometry

BACKGROUND: The clearance of D+ red blood cells (RBCs) from the circulation in D- individuals mediated by passively administered anti-D occurs by opsonization with the antibody and subsequent removal in the spleen. Few data exist on the kinetics of clearance of large volumes of D+ RBCs from the maternal circulation by anti-D in clinical cases of massive fetomaternal hemorrhage (FMH). CASE REPORT: A 33-year-old D- woman delivered a D+ female infant by emergency cesarean section for suspected fetal anemia. A massive FMH, initially estimated to be approximately 142 mL of RBCs, was found. In addition to the standard dose of intramuscular (IM) anti-D (300 microg) given immediately after delivery, 2700 microg of anti-D was administered intravenously (IV). The clearance of D+ fetal cells from the maternal circulation was monitored by flow cytometry in samples obtained on a daily basis using anti-D. The mother had no detectable anti-D 6 months after delivery. RESULTS: No clearance of fetal cells was apparent after the insufficient dose of IM anti-D. The IV administration of anti-D caused accelerated clearance of D+ fetal RBCs with a t1/2 of 24.5 hours. D+ reticulocytes comprised 4.2 percent of all D+ cells in the maternal circulation at delivery suggesting acute fetal blood loss. CONCLUSIONS: The approach used in this report allowed a detailed analysis of the kinetics related to the clearance of fetal D+ RBCs. Simultaneous measurements of fetal reticulocytes and fetal RBCs in maternal blood may establish the timing of an FMH.     

Transfusion of fresh murine red blood cells reverses adverse effects of older stored red blood cells

BACKGROUND: Although a subset of recent studies have suggested that red blood cell (RBC) storage length is associated with adverse patient outcomes, others have shown no such relationship. Adults may be transfused with RBC units of different storage lengths, and existing studies do not take into consideration that fresh RBCs may alter responses to concurrently transfused stored RBCs. To test this possibility, we utilized a murine model and investigated transfusion outcomes of fresh, stored, or fresh‐plus‐stored RBCs. STUDY DESIGN AND METHODS: Fresh, 14‐day‐stored or fresh plus 14‐day‐stored leukoreduced RBCs from HOD‐transgenic donors (with RBC‐specific expression of hen egg lysozyme, ovalbumin, and human Duffy^b) were transfused into naïve C57BL/6 recipients. Serum cytokines and anti‐HOD alloimmunization were evaluated after transfusion. RESULTS: In six of six experiments (n = 90 mice total), a proinflammatory serum cytokine storm of interleukin‐6, keratinocyte‐derived chemokine/CXCL1, and monocyte chemoattractant protein‐1 was observed in transfusion recipients of stored but not fresh RBCs, along with high degrees of anti‐HOD alloimmunization. However, concurrent transfusion of fresh HOD RBCs along with stored HOD RBCs significantly decreased these adverse outcomes (p < 0.05). CONCLUSIONS: These results are consistent with fresh murine HOD RBCs losing protective properties during storage, and introduce a previously unrecognized variable in RBC storage studies. If translatable to humans, uniform “old blood” groups may be needed in future clinical studies to more accurately investigate the biologic effects of older RBC units.     

Compatibility test using 51chromium-labeled red blood cells in crossmatch positive patients

For the past three years, we have routinely used the one-hour in vivo survival of ⁵¹Cr-labeled donor red blood cells to select units for transfusion to patients for whom crossmatch compatible blood was unavailable. This technique successfully evaluated in vivo compatibility, thus avoiding acute hemolytic transfusion reactions in 38 problem patients. New or confirmatory data regarding the hemolytic potential of several well defined antibodies was also obtained. Antibodies which proved to be clinically insignificant included: anti-I^T (all IgG), anti-Sd^a, anti-Kir, Mil, Oca, anti-Chido, anti-Bg, 14 of 15 “nonspecific warm autoantibodies” (three of which were associated with the ingestion of alphamethyldopa), and four of five antibodies to high-incidence antigens. Clinically significant antibodies included: anti-Yt^a, anti-Jk^b, the antibody in PCH (with “P” specificity), one intensely hemolytic “nonspecific warm autoagglutinin,” and one of five incompletely characterized antibodies to high-incidence antigens. An acceptable in vivo compatibility test in every instance was associated with an appropriate rise in hematocrit and no clinical symptoms of hemolytic transfusion reaction.     

Inactivation of protozoan parasites in red blood cells using INACTINE PEN110 chemistry

BACKGROUND: The transmission of parasites, including Babesia, plasmodia, and Trypanosoma cruzi, via transfusions is an important public health concern. INACTINE technology is a pathogen‐reduction process that utilizes PEN110, an electrophilic agent that inac‐tivates a wide range of pathogens by disrupting nucleic acid replication. The present study investigated the effect of PEN110 treatment on the viability of protozoa in RBCs. STUDY DESIGN AND METHODS: B. microti‐parasitized RBCs from infected hamsters were treated with PEN110 and inoculated to naïve animals. Parasitemia was detected by blood smears and PCR. Human RBCs infected with P. falciparum were treated with PEN110 and incubated with fresh RBCs. P. falciparum multiplication was detected by blood smears. Human RBCs spiked with T. cruzi and treated with PEN110 were analyzed for the presence of live parasites using in‐vitro infectivity assay or by inoculating susceptible mice. RESULTS: Treatment of RBCs infected with B. microti or P. falciparum with 0.01 to 0.1 percent (vol/vol) PEN110 resulted in parasite inactivation to below the limit of detection during 24 hours. T. cruzi inoculated into human RBCs was inactivated below the limit of detection by 0.1 percent PEN110 after 3 hours. CONCLUSION: The study demonstrates that treatment of blood with PEN110 is highly effective in eradicating transfusion‐transmitted protozoan parasites.     

Elution of antibody from red blood cells using xylene—a superior method

Chan-Shu and Blair's findings that xylene produces a strongly reactive eluate from red blood cells have been confirmed and extended. Alloantibodies of 21 different blood group specificities and red blood cells sensitized in vivo, including ABO sensitization of babies' blood, were tested. The xylene method produced stronger reacting eluates than the modified ether method in 70 per cent of the tests. Ether, acid, and heat methods never produced significantly stronger eluates than did xylene. Ether eluates prepared from red blood cells sensitized with anti-S and anti-s were nonreactive, but the same cells yielded easily detectable anti-S and anti-s when xylene eluates were prepared. The xylene method is sensitive and simple. In addition, xylene is not subject to such stringent control of storage conditions by inspection agencies as is ether.