Transfusion-associated microchimerism.

Blood transfusion is a newly recognized cause of microchimerism, the stable persistence of a minor population of allogeneic cells. Relatively recent advances in polymerase chain reaction technology have spawned new information about the frequency and aetiology of transfusion-associated microchimerism (TA-MC). Although conceptually related to fetal-maternal microchimerism, TA-MC is a distinct and separate entity. Evidence of TA-MC has been strongest among patients with severe traumatic injuries who receive relatively fresh blood products shortly after an episode of massive haemorrhage. The presence of a focal deficit in the cellular immunologic repertoire prior to transfusion that happens to match a blood donor's human leucocyte antigen type also appears to be an important predisposing factor. TA-MC seems to be common (affecting approximately 10% of transfused injured patients), enduring (lasting years to decades) and pronounced (involving up to 5% of circulating leucocytes and multiple immunophenotypic lineages suggestive of haematopoietic engraftment). Further study of this topic may reveal important information regarding potential clinical consequences of TA-MC, as well as basic haematologic and immunologic processes.

Real-time monitoring of enzymatic hydrolysis of galactomannans.

Enzymatic hydrolysis was monitored in real-time using time dependent static light scattering (TDSLS) for a variety of galactomannans from native Brazilian flora. alpha-Galactosidase, which strips only the (1-6)alpha-D galactose side groups, and beta-mannanase, which hydrolyses only the (1-4)beta-D mannan main chain into oligosaccharides were investigated separately and in combination. The time-dependent signatures matched those describing side-chain stripping for galactosidase, whereas those resulting from the action of mannanase followed the signature typical of random backbone cleavage. Use of both enzymes together required that the TDSLS theory of polymer degradation be extended to the case where random backbone cleavage sites appear as side chains are stripped by the first enzyme. Whereas galactosidase allowed mannanase to access more backbone cleavage sites as time passes, leading to a higher degree of hydrolysis, there was no increase in rate constants. The distribution of random fragments in the case of mannanase digestion alone followed reasonably well the predictions for random cleavage of a single-strand polymer with a restricted number of cleavage sites. The fragment distributions were evaluated by size exclusion chromatography.

Detection of microchimerism by PCR is a function of amplification strategy.

BACKGROUND: Suitable detection methods are needed to support larger studies of microchimerism and the allogeneic exposures that may be etiologically related to it. STUDY DESIGN AND METHODS: A twotier PCR strategy for microchimerism detection was developed on the basis of the observation that assay sensitivity for the detection of microchimerism depends on the specificity with which primer pairs recognize sequences unique to the minor population. First, specimens are tested to determine the host HLA class II genotype by using a locus-specific PCR strategy with low sensitivity for microchimerism. Then, a sequence-specific PCR analysis having high sensitivity for detection of microchimerism is applied to detect and quantitate the minor population. Locus-specific, group-specific, and sequence-specific amplification strategies for the detection of distinct minor WBC populations prepared ex vivo were compared. In addition, 39 clinical samples from patients with known transfusion-associated microchimerism and 20 umbilical cord blood (CB) specimens containing maternal WBCs were studied. RESULTS: Locus-specific amplification detected 17 (94%) of 18 cases in which microchimerism was present at 10 percent, but only 1 of 51 cases with microchimerism < or = 1 percent. Group-specific amplification detected all 63 cases with minor populations present at > or = 0.10 percent, but only 16 of 21 cases at the 0.01 percent level. Sequence-specific amplification detected 100 percent of cases down to the 0.01 percent level. When applied to clinical samples, locus-specific amplification reliably identified the major population but proved insensitive to low-level minor populations. CONCLUSIONS: For the detection of microchimerism, assay sensitivity is a function of amplification strategy. These results suggest a simple approach to population screening for microchimerism: the background population of WBCs is typed by a locus-specific method, while minor population(s) can then be sought by using one or several sequence-specific amplifications.

New evidence of the nonequilibrium nature of the "slow modes" of diffusion in polyelectrolyte solutions.

The time-dependent behavior of the dissolution of polyelectrolyte powders in pure water and moderate ionic strength aqueous solvent was monitored by flowing dissolving material through an online filter, and then through a multiangle light scattering unit, a refractometer, and a capillary viscometer. When the polyelectrolytes were dissolved in solutions of moderate ionic strength, their dissolution behavior was similar to that of neutral polymers. When dissolved in pure water, however, there was consistently a small population of aggregates that appeared at the beginning of the dissolution process, which then rapidly diminished. For large pore filtration, the aggregates reached a final low level, and slowly disappeared over the span of many days, whereas for small pore filtration the aggregates disappeared completely over a scale of minutes. The real-time data, together with size exclusion chromatography analysis, shed light on previously unanswered questions concerning the nonequilibrium nature of this small population of polyelectrolyte aggregates in low ionic strength solutions, and its relation to the "extraordinary phase" of diffusion (or "slow modes"). Further evidence is also provided that both angular scattering maxima due to interpolyion correlations and the maximum of reduced viscosity vs polyion concentration ("electroviscous" effect) at low ionic strength are equilibrium properties that are unrelated to these aggregates.

Transfusion practice for patients with sickle cell disease.

The practice of transfusion for patients with sickle cell disease is changing. Diminished childhood mortality is resulting in greater patient longevity and a higher prevalence of chronic systemic complications. Many of these chronic complications, as well as several acute ones, are treatable with erythrocyte transfusion. Proactive or preventive uses for transfusion are being considered for selected patients. Immunologic phenomena, hemosiderosis, and risk for transmission of infectious agents are the three most noticeable risks associated with repetitive transfusion. Evolving work with alternative oxygen carriers and surface modification of erythrocytes is also promising and relevant for patients with sickle cell disease.

Fluorescence-based test of fiber-optic continuity.

There is considerable interest in the use of lasers and optical fibers for the initiation of pyrotechnics. In this application the need develops for a means of testing the continuity of the initiation fiber before initiation of the pyrotechnic. We present proof of the feasibility of an unambiguous continuity test using the fluorescence returned by the fiber from a fluorescent material in or near the pyrotechnic.

Conformations and flexibility of native and re-natured xanthan in aqueous solutions.

The conformation and flexibility of sonicated 'native' and 're-natured' xanthan have been investigated by size exclusion chromatography (SEC) with coupled multi-angle light scattering and viscosity detectors. 'Native' xanthan (NX) refers to xanthan dissolved in moderate ionic strength aqueous solution, which has not been exposed either to high temperature or very low ionic strength, and 're-natured' xanthan (RX) here refers to xanthan which has been heated above the conformational melting temperature and then recooled. The mass distributions of the NX and RX are virtually identical, implying that the RX does not involve aggregates of, or disassociated fragments of, NX. The flexibilities and conformations between NX and RX, however, are strikingly different; RX is far stiffer than NX, the persistence lengths being roughly 1000 A and 300 A, respectively, and the mass per unit length M/L of the RX is roughly double that of NX. With estimated M/L of 200 Da/A and 98 Da/A, respectively, the results strengthen the notion that RX is double stranded, whereas as NX appears single stranded. The nature and mechanism of formation of the double-stranded form is still unclear, and a few speculative scenarios are suggested. Finally, preliminary results on the kinetics of xanthan self-association in HCI are presented which illustrate the complexity of such processes in xanthan.

High osmotic stress behavior of hyaluronate and heparin.

Using polyethylene glycol and dextran as osmotic stressing agents, the concentrations of hyaluronate and heparin were measured as a function of osmotic pressure II over the range of 0.03 to nearly 50 atmospheres. The experimental results were analyzed in terms of the Donnan osmotic pressure, the virial expansion, and Flory's first neighbor interaction parameter. In addition, II was looked at as a function of the reciprocal cube root of the concentration, which represents an average intermonomer spacing at high concentrations. The decay lengths in the so-called hydration region were found to be around 2.6 A and negligibly salt dependent. In the electrostatically dominated region the decay lengths were found to be dependent on the ionic strength, but not simply so. The osmotic compressibilities were also calculated, and were compared to compressibility data of corneal stroma and articular cartilage. These latter compressibilities were close to those for the pure hyaluronate and heparin, strengthening the evidence that glycosaminoglycans (GAGs) are largely responsible for connective tissue compressibility. Higher compressibilities for previously reported GAG data is thought to be related to the protein content of those samples.

Characterization and time dependence of amphotericin B: deoxycholate aggregation by quasielastic light scattering.

Quasielastic light scattering measurements of amphotericin B (AB):deoxycholate (DOC) preparations provided information about particle size and aggregation as a function of concentration. The data allowed the time dependence of the aggregation to be followed and indicated that the initial rates of the change in average equivalent hydrodynamic diameter increased with decreasing concentration. The results extend the model proposed by Lamy-Freund and co-workers, which describes AB:DOC systems as consisting of AB:DOC mixed aggregates co-existing with pure DOC micelles. Although the AB:DOC aggregates are unstable at all concentrations studied, the rate of aggregation increases by three orders of magnitude as the concentration is reduced from 20 mM (DOC concentration) to the concentration region of DOC micellization. These results are in agreement with the different distribution of AB and DOC in the body of experimental animals, and may be of relevance for the understanding of the serious toxic effects of AB.

Random coil scission rates determined by time-dependent total intensity light scattering: hyaluronate depolymerization by hyaluronidase.

A recently developed theory of the light scattering by random coils undergoing random scission is applied to the digestion of hyaluronate by hyaluronidase. The time dependence of the scattered light from solutions undergoing digestion was monitored. Working at a high angle with high molecular weight hyaluronate allowed the use of a powerful approximation for determining initial velocities and the Henri-Michaelis-menten coefficients, without explicit knowledge of the hyaluronate molecular weight, radius of gyration, second virial coefficient, or polydispersity. Effects due to a molecular weight dependent second virial coefficient and to non-Gaussian behavior are briefly considered. Assays were performed over nearly two orders of magnitude in substrate concentration. The initial velocities are compared with those obtained by a standard reducing sugar assay, which was performed on identical samples. The main advantages of the light scattering assay procedure over the more traditional assays are that many relatively high-precision data points can be quickly and automatically collected with simple apparatus, and that the technique is most sensitive for the initial period of digestion, where the other assays are least sensitive. The shapes of the scattering curves also provide evidence that hyaluronate in these solutions is not a stable double strand and that the hyaluronidase cleaves bond randomly. The curves also indicate that enzyme deactivation occurs, which accounts for the lower velocities yielded by the slower reductimetric assay, which is measured over longer initial periods.

The effects of pH on hyaluronate as observed by light scattering.

Hyaluronate was investigated over a wide pH range, and at near zero and intermediate ionic strength, using dynamic and total intensity light scattering. Commercially obtained rooster comb hyaluronate was purified, and solutions were prepared in pure water by low-power bath ultrasonication and subsequent filtering. These solutions were of low polydispersity and appeared to contain single molecules of hyaluronate. Despite the absence of added electrolyte, these solutions yielded well-behaved Zimm plots. Increasing ionic strength and changing pH decreased radii of gyration and increased diffusion constants. Except for what appeared to be slow hydrolysis at either extreme of pH, molecular weights remained constant under all pH and ionic strength conditions. Under all solvent conditions investigated, diffusion coefficients increased with decreasing hyaluronate concentration. Unsonicated, lightly centrifuged solutions without added electrolyte were polydisperse, and their light scattering intensity was dominated by what appeared to be stable hyaluronate aggregates. The results are interpreted in terms of the polyelectrolyte properties of hyaluronate and its tendency to form stable entanglements, especially at low ionic strength. Previous light scattering studies in the literature on hyaluronate have shown widely varying results. The present article briefly reviews this literature and attempts to explain the variation among the previous results, emphasizing the Kuhn statistical segment length as an indicator of whether results are influenced by polydispersity or contaminants causing hyaluronate aggregation.