Treasure of the Past VII: Measurement of the Thickness and Refractive Index of Very Thin Films and the Optical Properties of Surfaces by Ellipsometry.

The use of the ellipsometer for the measurement of the thickness and refractive index of very thin films is reviewed. The Poincaré sphere representation of the state of polarization of light is developed and used to describe the reflection process. Details of the operation of the ellipsometer are examined critically. A computational method is presented by which the thickness of a film of known refractive index on a reflecting substrate of known optical constants may be calculated directly from the ellipsometer readings. A method for computing both the refractive index and thickness of an unknown film is also developed. These methods have been applied to the determination of the thickness of an adsorbed water layer on chromium ferrotype plates and on gold surfaces. In the former case the thickness was 23 to 27 Å, and in the latter was 2 to 5 Å. The measurement of the thickness and refractive index of barium fluoride films evaporated on chromium ferrotype surfaces is used as an illustration of the simultaneous determination of these two quantities.

Distribution of HIV type 1 (HIV-1) in blood components: detection and significance of high levels of HIV-1 associated with platelets.

BACKGROUND: Although inactivation of enveloped viruses transmitted by plasma derivatives has been successful, no methods for virus inactivation or removal have been established for platelet concentrates or red cell (RBC) components. Relatively little is known regarding the extent or significance of virus interactions with the cellular constituents in these components. STUDY DESIGN AND METHODS: Units of whole blood were collected from six HIV type 1 (HIV-1)-positive, asymptomatic individuals and separated into peripheral blood mononuclear cells (PBMNCs), cell-free plasma, white cell-reduced platelet concentrate, and white cell-reduced RBCs. DNA and RNA polymerase chain reaction and virus culture methods were used to study the compartmentalization of HIV-1 immediately after component preparation and after storage. RESULTS: As expected, HIV DNA and infectious virus were detected in fresh blood and in PBMNCs, and virion-associated RNA was detected in fresh plasma from all six donors. The levels of viral nucleic acids in these preparations remained relatively stable with 4 degrees C storage, whereas infectivity of PBMNCs was rapidly lost. Washed RBCs tested negative for HIV in all assays at all time points. Platelets retained high levels of HIV RNA (but not infectivity) after extensive washing, as well as after storage at 4 and 22 degrees C. High-level platelet-associated HIV-1 was also demonstrated in samples collected during early seroconversion. Periseroconversion and postseroconversion levels of platelet-associated HIV-1 correlated with the level of plasma viremia and with the rate of progression to AIDS. Cell-free virus from donor plasma and tissue culture fluid rapidly and firmly attached to platelets from noninfected donors. Infectivity of tissue culture virus bound to platelets was demonstrated in vitro. CONCLUSION: Significant levels of HIV-1 are associated with platelets during all stages of infection. Platelet-associated HIV could either mediate virus clearance or facilitate virus dissemination and expanded tropism. Finally, virus inactivation research must address virus associations with platelets.

Quality control of white cell-reduced red cells: white cell preservation and simplified counting.

BACKGROUND: White cell (WBC) degradation restricts the interval between the filtration process and the assay for residual WBCs. Maintaining WBC integrity would permit extended sample storage for batching and/or shipment to centralized laboratories. The usual quality control assay for WBC-reduced red cell units requires determining the number of WBCs in the entire counting area of a Nageotte hemocytometer, which consists of 40 rows. Reducing the counting area would simplify the quality control procedure. STUDY DESIGN AND METHODS: Adsol red cell units were prepared either on the day of collection (Day 0) or on Day 1 and WBC reduced by filtration on the same day. By using prefiltration and postfiltration red cells, samples containing WBC concentrations of 15, 10, and 3 WBCs per microL were prepared by serial dilution. Identical samples were treated with glutaraldehyde and stored at either 20 to 24 degrees C or 1 to 6 degrees C. All samples were assayed on the day of component preparation and on Days 7 and 14. The numbers of WBCs corresponding to 10- and 40-row areas of the Nageotte hemocytometer were determined. RESULTS: For the conditions and WBC concentration range studied, no significant changes in WBC concentrations were observed through Day 14 for glutaraldehyde-treated samples stored at either temperature, although there were substantial decreases in untreated samples. A 10-row measurement was determined to be sufficient for identifying WBC-reduced red cell units passing the present limit of 5 x 10(6) residual WBCs. CONCLUSION: Glutaraldehyde treatment can preserve WBCs in red cell samples at least up to Day 14, which provides increased efficiency in quality control for laboratories. Current red cell WBC-reduction filters produce components that, when assayed, contain fewer than 10 WBCs per full counting area. The simplified procedure would allow reduction of the counting area by 75 percent.

Evaluation of a new prestorage leukoreduction filter for red blood cell units.

BACKGROUND AND OBJECTIVES: Prestorage leukoreduction offers a variety of potential benefits and is becoming more commonly practiced. The LeukoNet prestorage leukoreduction filtration system is intended for leukoreduction of red blood cells and uses a vent to allow automatic drainage of red cells from the filter. MATERIALS AND METHODS: We studied the functional characteristics and the in-vivo and in-vitro properties of leukoreduced AS-1 Red Blood Cells prepared with this new system. Units of AS-1 Red Blood Cells were filtered at 4 degrees C through the LeukoNet filter 24-48 h after collection and stored under usual conditions for 42 days. Residual leukocytes were enumerated using a Nageotte chamber or with a polymerase chain reaction (PCR) technique. In the clinical trial (phase one), 21 donors had units stored with and without leukoreduction for 42 days; biochemical assays were done before and after storage, and 51Cr/99mTc red cell recovery studies at the end of the storage period. RESULTS: Leukocyte content after filtration was 3.2 +/- 2.6 x 10(4)/unit (n = 21), and all units had < 1 x 10(5) leukocytes (median: 3.8 x 10(4)). In-vivo paired studies showed no difference in 24-hour recovery (control: 82.1 +/- 5.8%; test: 82.9 +/- 6.0%). Hemolysis was halved with leuko-reduction (0.59 +/- 0.30 vs. 0.29 +/- 0.11%; p < 0.05), and glucose consumption was reduced by 5% compared to control units (p = < 0.05). Other biochemical parameters showed no differences. In the practical trial (phase two), filtration time was 41 +/- 23 min. With a residual leukocyte content of 6.6 +/- 4.9 x 10(4)/unit and 14 +/- 3% red cell loss (n = 84). Six additional units underwent leukocyte enumeration by PCR and had 2.6 +/- 1.1 x 10(4) residual leukocytes. CONCLUSIONS: Under the conditions studied, the LeukoNet leukoreduction filtration system produces about 4-5 log10 leukocyte content reduction.

Multicenter evaluation of the 3% paraformaldehyde method for white cell counting in leukocyte-reduced red blood cells. BEST (Biomedical Excellence for Safer Transfusion) Working Party of the International Society of Blood Transfusion.

BACKGROUND/AIM: The 3% paraformaldehyde (PFA) method is a simple technique for counting residual white blood cells (WBC) in leukocyte-depleted red blood cells (RBC). Preliminary data suggested that its sensitivity is at least equal to PCR and flow cytometry. We report the results of a multicenter study conducted by the BEST Working Party to determine precision and accuracy of the 3% PFA method. STUDY DESIGN: In the 7 participating laboratories, 5 sets of samples containing nominal concentrations of 200, 100, 50, and 10 WBC/ml were prepared by diluting whole blood into 'WBC-free' RBC. Ten milliliters of each sample were processed using the 3% PFA method, which is based on erythrocyte lysis and WBC concentration into 5% of the original sample volume; a Nageotte chamber is used to count concentrated WBC. RESULTS: The precision of the technique varied according to the nominal concentration, ranging from a CV of 12% at 200 WBC/ml to 57% at 10 WBC/ml. The technique measured fewer than the nominal WBC concentrations (mean of all laboratories, -12.4%); underestimation was probably due to cell loss during sample manipulation. Overall accuracy was however acceptable, because statistical considerations establish that the actual WBC concentration would unlikely exceed 2 times the estimated count. CONCLUSIONS: The 3% PFA method is suitable for the enumeration of residual WBC at concentrations > or = 50/ml. It represents a useful tool for evaluation of high performance filters by reference laboratories.

In vitro and in vivo effects of prestorage filtration of apheresis platelets.

BACKGROUND: The effect of prestorage filtration on the quality of apheresis platelet concentrates stored for transfusion is undetermined. STUDY DESIGN AND METHODS: Investigation of 11 plateletpheresis components used a concurrent paired-study design. On the day of collection, each component was equally divided into two suspensions; one half was filtered, and the other half was not. Each suspension was stored for 5 days. In vitro testing was performed on the day of collection (Day 0) for cell counts and on Day 5 for measurements of lactate, glucose, blood gases, pH, platelet ATP, hypotonic stress ratio, extent of shape change in response to ADP, tissue necrosis factor alpha, interleukin 8, interleukin 1 alpha, interleukin 1 beta, interleukin 6, and platelet surface glycoproteins by flow cytometry. At the end of the 5-day period, a sample was taken from each of the two suspensions, radiolabeled with either 51Cr or 111In, and transfused concurrently. Posttransfusion samples were drawn for measurements of recovery and platelet survival and for functional assessment of the ex vivo ability of the circulating radiolabeled platelets to aggregate in response to ADP. RESULTS: The apheresis component had a mean platelet yield of 3.2 +/- 0.4 x 10(11) and a white cell yield ranging from 1 x 10(5) to 1 x 10(8), with a median of 2 x 10(7). Filtration resulted in a platelet loss of approximately 10 percent and a variable 2 to 3 log10 reduction in white cell content. No significant differences between filtered and unfiltered suspensions in paired t tests that would likely have an impact on platelet quality were observed in the in vitro tests. The in vivo recovery and survival were highly similar and not statistically different in filtered and unfiltered paired suspensions: the mean difference was 1.2 +/- 4.0 percent for recovery and 7.0 +/- 15 hours for survival. The functional assessment by aggregation to ADP showed no difference between filtered and unfiltered suspensions. A small decrease in tumor necrosis factor alpha and interleukin 8 was evident in the filtered suspension as compared to levels in the unfiltered suspensions. CONCLUSION: Prestorage white cell reduction in apheresis components resulted in WBC reduction by several log10 with no evident adverse effect on platelet viability or function.

Reducing the infectivity of blood components--what we have learned.

The safety of the nation's blood supply has improved over the last several years as a result of more intensive donor screening and viral testing. Concurrently, there has been more judicious use of blood components. Although the risk is small, transmission of blood borne viruses, bacteria and parasites can occur. Investigators have studied a myriad of processes for pathogen depletion and/or inactivation, including the use of chemicals, extended storage, filtration, heating, irradiation, photochemicals and washing. Pasteurization, methylene blue and solvent-detergent processes have been introduced in parts of Europe for improving the safety of plasma used for transfusion. The FDA is reviewing a license application for the solvent-detergent process. For red cells, use of highly efficient leukodepletion filters is believed to be equivalent to antibody testing for the prevention of CMV disease transmission. Otherwise, no successful treatments have yet been identified for red cells or platelets. Several photochemicals, which may be useful for treating these components, are being studied. However, there appear to be trade-offs between the extent of pathogen inactivation, platelet or red cell damage, and genotoxicity. These as well as other biological parameters and operational issues will need to be further evaluated before implementation can be considered.

Quantitation of residual white cells in filtered blood components by polymerase chain reaction amplification of HLA DQ-A DNA.

BACKGROUND: Over the past several years, blood filtration technology has improved dramatically, such that currently available experimental filters are capable of reducing white cells (WBCs) in blood components to less than 0.1 WBC per microL. These residual WBC concentrations are below the sensitivity of automated cell counters, as well as of large-volume (Nageotte) hemocytometers. STUDY DESIGN AND METHODS: A quantitative polymerase chain reaction (PCR) amplification assay directed at HLA DQ-A DNA sequences has been developed for the enumeration of WBCs in filtered blood. To ensure quantitative recovery of WBCs at very low residual cell concentrations, a direct red cell lysis and WBC concentration protocol using 0.5 mL of filtered blood was perfected. Amplified product is detected by oligomer hybridization using 32P-labeled probes, with quantitation by image analysis of autoradiographic signals relative to a standardized dilution series processed in parallel. RESULTS: Recovery of residual WBCs in filtrates was shown to be enhanced by the addition of xenogeneic WBCs or polystyrene beads, which served as "carrier" particles during red cell lysis and wash steps. A contribution of nuclear fragments in filtered blood to PCR signal in the range of 0.01 to 0.5 WBCs per microL was observed; a modified protocol was developed to minimize this effect. Parallel analysis of spiked dilution series and evaluations of 39 red cell components filtered through commercial filters indicated good correlation between PCR and standard Nageotte counts in the range of 0.1 to 10 WBCs per microL (r2 = 0.94); only PCR was able to detect residual WBCs in filtrates from prototype 6 log10 WBC-reduction filters. CONCLUSION: This assay should prove useful for the development and quality assurance of increasingly efficient WBC-reduction filters.

Loss of red blood cell viability associated with limited thermal inactivation of extracellular HIV-1.

The effects of incubation at mildly elevated temperatures on HIV-1 inactivation and in vitro red blood cell properties were investigated. Red cells (55% Hct) were leukodepleted (3 log10) by filtration, maintained at 45 or 47 degrees C for 4 or 8 h, and then stored at 4 degrees C. Hemolysis was twice that of controls after 42-day storage for samples treated for 4 h at 45 degrees C, and five times larger for samples heated at 47 degrees C. There was also a significant increase in the rate of potassium loss, an early decrease in ATP levels, and an initial drop in pH for samples treated at either temperature. Larger differences were observed for samples exposed to these elevated temperatures for 8 h. Osmotic deformability curves obtained by ektacytometry showed dramatic decreases in red cell deformability at both temperatures and for both time periods. HIV-1 inactivation in red cells treated at 45 degrees C (approximately 0.25 log10/h) was considerably less than that obtained in tissue culture medium (1-2 log10/h). Since the decrease in red cell deformability is likely to indicate reduced red cell function and survival, and the rate of HIV-1 inactivation is low, mild heat treatment is not an adequate process for viral inactivation of red cell products.

Red cell alterations associated with virucidal methylene blue phototreatment.

Several red cell storage properties were evaluated following phototreatment with methylene blue (MB) under conditions that inactivated > or = 6 log10 of added vesicular stomatitis virus. Red cell 2,3 DPG levels were similar to untreated controls throughout conventional 42-day storage at 4 degrees C. Plasma hemoglobin levels were elevated approximately twofold in MB-phototreated samples, and morphology scores were 5 percent lower after 42-day storage. ATP levels declined 30 percent in phototreated samples and in a control sample containing MB and not exposed to light. Lipid peroxidation was not observed in treated or control cells, nor were differences observed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis of ghost membranes derived from phototreated and control samples. Phototreated cells exhibited enhanced ion permeability; sodium and potassium levels approached equilibrium with the suspending medium within 4 to 7 days after treatment. Direct agglutination tests using rabbit anti-human IgG or rabbit anti-human serum albumin on MB-phototreated cells indicated that serum proteins had absorbed to the surface of treated red cells. Plasma depletion by washing red cells prior to phototreatment did not prevent protein binding upon subsequent addition of untreated autologous or group AB plasma. To a much smaller extent, phototreatment of plasma resulted in IgG association with untreated red cells. The addition of glutathione to red cell suspensions prevented IgG binding to phototreated red cells but did not prevent enhanced ion permeability. Taken together, these data suggest that the red cell surface is altered by virucidal MB phototreatment of vesicular stomatitis virus.

Viral inactivation and reduction in cellular blood products.

Even though the risks associated with the transfusion of blood products are lower than ever before, considerable efforts are being employed to improve the safety of the blood supply. Based upon available data, a six log (99.9999%) reduction in virus level from screened and tested blood components should significantly reduce or eliminate the risk of post-transfusion infection. The objective has been to identify "generic" methods, that is, one that would be applicable to all virus. For red cells, physical and chemical approaches have been studied; for platelets, the approaches have been limited to chemical. The physical methods include depletion of leukocytes by filtration, removal of plasma by washing, and viral inactivation by heat. Among the chemicals investigated to inactivate or help displace virus are ozone, detergents, and hypochlorous acid. Several photochemicals have also received intensive investigation: merocyanine 540, a benzoporphyrin derivative, aluminum phthalocyanine, and methylene blue. For platelets, photochemical inactivation methods using merocyanine 540, and two psoralen derivatives, 8-methoxypsoralen (8-MOP) and aminomethyl trimethyl psoralen (AMT), have also been studied. Approaches which include washing are not suitable. For the most part, either viral removal or inactivation has been insufficient, or red cell or platelet damage unacceptable. However, there are a few indications that at least inactivation of a specific virus, such as HIV, may be possible without major cell damage. These studies are in their early stages and significant work remains. If feasibility is clearly shown in vitro, it is likely that in vivo primate studies to demonstrate safety and efficacy will be required.

Experimental 6 log10 white cell-reduction filters for red cells.

White cell (WBC) reduction of blood components has been receiving increased attention as a way of reducing transfusion-related complications such as WBC-associated HLA alloimmunization and transmission of cell-associated viral diseases. Currently available filters are limited to removing approximately 3 log10 (99.9%) of WBCs from red cells (RBCs). The performance of two experimental filters that were designed to remove 6 log10 WBCs from fresh RBCs during component preparation was evaluated. Both filters were able to meet this objective in less than 40 minutes with RBC losses of less than 15 percent under nonoptimized conditions. Filtered RBCs showed storage parameters within the normal range over a 42-day period. The use of these filters, if combined with a sterile docking device or if incorporated into a collection set, should provide the means to supply highly WBC-reduced RBCs with a normal shelf life.

Methods for measuring a 6 log10 white cell depletion in red cells.

Methodology is presented for enumerating very low concentrations of white cells (WBCs) in red cells (RBCs) by two separate measurement techniques. Both techniques rely on the method of harvesting WBCs from a 300- to 350-mL unit of RBCs and concentrating them to a volume of approximately 0.5 to 1.0 mL, which is equivalent to a WBC concentration of approximately 550 to 1. The WBC separation and concentration steps require less than 3 hours to complete, and multiple RBC units can be processed in parallel. Cell counting is carried out in a fluorescence hemocytometer or by a modified cytospin technique. As few as 1000 WBCs in a unit of RBCs, which corresponds to a more than 6 log10 WBC depletion, can be measured without reaching the sensitivity limit of either technique (800 and 200 WBC/unit, respectively). The harvesting method and counting techniques are relatively simple and inexpensive.

The effect of flow on the interaction of isolated megakaryocytes with subendothelial extracellular matrix.

We have previously shown that human, guinea pig, or rat megakaryocytes, incubated under static conditions on an extracellular matrix (ECM) produced by endothelial cells, readily adhered to the matrix and underwent platelet-like shape change and thromboxane A2 secretion. We have now exposed megakaryocytes to ECM in a perfusion system similar to that used to study platelets circulated over aortic subendothelium. We used a continuous flow circuit incorporating a parallel plate perfusion chamber. Megakaryocytes were isolated to high purity from guinea pig marrow by centrifugal elutriation and velocity sedimentation. The cells were introduced into the flowing medium while the surface of an ECM-coated coverslip mounted in the chamber was observed continuously by phase-contrast video microscopy for up to 18 hours. Megakaryocytes from the flowing suspension started to adhere to the ECM within seconds. Significant adhesion occurred over a range of shear rates, from 10 to 190 seconds-1, did not appear above 300 seconds-1 and was greatest at a shear rate of 60 seconds-1. Adhesion to the ECM was specific, since there was no adherence to glass coverslips, glutaraldehyde-fixed ECM-coated coverslips, or to endothelial cells cultured on ECM-coated coverslips. At low shear rates large aggregates of megakaryocytes formed on the ECM surface; these could be detached and washed away by higher shear forces. Megakaryocytes thus acquire, even before platelet formation, an adhesive capacity similar to that of platelets. In addition, a significant fraction of the adherent megakaryocytes underwent elongation and pseudopod formation similar to that seen in marrow sinusoids.

A flow system for the study of shear forces upon cultured endothelial cells.

A parallel plate chamber in a flow system has been designed to study the effects of fluid shear stresses on cells. The system was applied to the study of cultured endothelial cells grown on cover slips which were accommodated in recessed wells in the base plate. Dye injection studies in the chamber indicated laminar flow over the cells. Shear rates measured over the cover slips by an electrochemical technique were found to be linear with flow rate. Laser doppler anemometry showed parabolic profiles between the plates. Endothelial cells subjected to flow showed a correlation between the time required for orientation and the magnitude of the shear stress.

Enhanced production of human gamma-interferon in nylon column-fractionated cell cultures.

The production of human gamma-interferon (HuIFN-gamma) in unfractionated and nylon wool column-fractionated leukocyte cell cultures stimulated with PMA and PHA was investigated. Production was studied with normal and reduced autologous serum protein levels in 96-hr spinner cultures. A 10- to 15-fold enhancement of production and a 50-fold increase in specific activity of crude HuIFN-gamma was demonstrated in nylon column-fractionated/reduced serum cell cultures. Kinetic analysis revealed a production rate maximum within 6 hr of induction in unprocessed cell cultures, whereas production occurred at an essentially constant rate for 48 hr in fractionated cell cultures.

Evaluation of membranes for plasmapheresis.

Commercial flat-sheet microporous membranes were evaluated for potential use in plasmapheresis with a specially designed filtration module. Significant differences in filtration rates were observed with different membranes. Saline filtration data were not useful in predicting the capacity of the membranes to filter plasma from whole blood. For all membranes studied, no rejection of plasma proteins was detected. No activation or deactivation of clotting factors was detected as a result of filtration. In addition, little or no hemolysis was caused by filtration with the various membranes. Saline, cell-free plasma, platelet-poor plasma, and whole blood were perfused over a track-etched membrane and the resulting filtration rates were compared. The cell-free plasma filtration rate decreased significantly with time, probably owing primarily to protein adsorption in the membrane pores. Cell-free plasma and saline filtration data were used to calculate an apparent adsorbed layer thickness in the membrane pores. Perfusion of platelet-poor plasma and whole blood resulted in time-dependent filtration rates that were much lower than those obtained when cell-free plasma was perfused. Results of the study support recent theoretical models that postulate that the rate-limiting process for blood filtration is the formation of a layer of blood cells (particle polarization) on the membrane surface.