A microfluidics approach for the isolation of nucleated red blood cells (NRBCs) from the peripheral blood of pregnant women.

OBJECTIVE: Nucleated red blood cells (NRBCs) have been identified in maternal circulation and potentially provide a resource for the monitoring and diagnosis of maternal, fetal, and neonatal health and disease. Past strategies used to isolate and enrich for NRBCs are limited to complex approaches that result in low recovery and less than optimal cell purity. Here we report the development of a high-throughput and highly efficient microfluidic device for isolating rare NRBCs from maternal blood. MATERIAL AND METHODS: NRBCs were isolated from the peripheral blood of 58 pregnant women using a microfluidic process that consists of a microfluidic chip for size-based cell separation and a magnetic device for hemoglobin-based cell isolation. RESULTS: The microfluidic-magnetic combination removes nontarget red blood cells and white blood cells at a very high efficiency (approximately 99.99%). The device successfully identified NRBCs from the peripheral blood of 58/58 pre-termination samples with a mean of 37.44 NRBC/mL (range 0.37-274.36 NRBC/mL). These results were compared with those from previous studies. CONCLUSION: The microfluidic device results in an approximate 10- to 20-fold enrichment of NRBCs over methods described previously. The reliability of isolation and the purity of the NRBC product have the potential to enable the subsequent application of molecular diagnostic assays.

Physicochemical assessments of parenteral lipid emulsions: light obscuration versus laser diffraction.

The United States Pharmacopeia (USP) has proposed a new Chapter <729> entitled 'Globule Size Distribution in Intravenous Emulsions' that is intended to identify methods for analyzing the stability of lipid emulsions. We studied the differences between particle-sizing instruments when analyzing the physicochemical stability of a parenteral nutrition mixture compounded with intravenous lipid emulsion, known as an all-in-one mixture. As the growth of lipid droplets, i.e. coalescence, signals an irreversible change in emulsion stability, we focused our investigation on the large diameter tail (>5 microm) of the globule size distribution. Of the four proposed methods, droplet size was studied over a range of mixture stabilities using a low osmolality parenteral nutrition formula employing both light scattering and light obscuration techniques. In addition, the same mixtures were also freshly prepared, and then spiked with a known amount of 5 microm latex spheres. The response obtained from the light obscuration technique was linear and detected both unstable and latex-spiked mixtures in every case for droplets or particles >5 microm. The results of the laser diffraction method were non-linear and overestimated, was less sensitive or missed entirely, globules or particles in the large diameter tail of the dispersion. The results demonstrate that light obscuration is superior to laser diffraction in identifying unstable intravenous fat emulsions.

Selective associations in day-old chicks: when do CS traces become available for sickness-conditioned learning?

Barber and colleagues (T. A. Barber, A. M. Klunk, P. D. Howorth, M. F. Pearlman, & K. E. Patrick, 1998; T. A. Barber, M. F. Pearlman & K. E. Patrick, 1995) showed that male domestic chicks would not peck a bead presented 15 or more min before a lithium chloride (LiCl) injection, but would peck a bead presented less than 15 min before a LiCl injection. In 4 experiments, the authors (a) confirmed their initial observation, (b) showed that the effect is not due to retrograde amnesia produced by LiCl, and (c) confirmed that memories less than 15 min old are available for other types of learning.

Lesions of the intermediate medial hyperstriatum ventrale impair sickness-conditioned learning in day-old chicks.

Lesion studies show that the intermediate medial hyperstriatum ventrale (IMHV), a forebrain visual association area in chicks, is involved in learning and memory for one-trial passive avoidance and imprinting. We examined the effects of IMHV lesions in a one-trial, nongustatory, sickness-conditioned learning task. This task is similar to passive avoidance and imprinting because all three tasks require the chick to remember visual cues in order to respond correctly. However, sickness-conditioned learning differs from imprinting and passive avoidance because it uses sickness as the aversive stimulus and there is a longer conditioned stimulus-unconditioned stimulus interval (30-min delay compared to seconds). Bilateral IMHV lesions given 24 h before training impaired the ability of the chicks to avoid a bead associated with sickness produced by lithium chloride injection, as did pretraining unilateral left or right IMHV lesions. Post-training IMHV lesions given 1 h after training did not impair avoidance of the test bead in the sickness-conditioned learning task. However, lesioned chicks showed generalized avoidance of all test beads. The pretraining lesion results are similar to those found in imprinting and passive avoidance learning; however, the effects of unilateral IMHV lesions differed. Post-training lesion effects are similar to those found in passive avoidance learning. We propose that both left and right IMHV are necessary for sickness-conditioned learning and that post-training IMHV lesions impair the ability of the chick to learn or remember the association between the color of the bead and the aversive consequences of LiCl injection.

A new look at an old task: advantages and uses of sickness-conditioned learning in day-old chicks.

In sickness-conditioned learning, animals become ill after sampling a new substance and develop an aversion that is expressed as avoidance of that substance in subsequent presentations. We examined the parameters of a one-trial, nongustatory, sickness-conditioned learning task in day-old chicks. Chicks pecked a bead and were made ill by i.p. injection of lithium chloride (LiCl). Both 0.5 and 1.0 M LiCl (0.1 ml) produced reliable avoidance at test. Chicks injected with LiCl between 15 and 45 min after training avoided the bead at test, whereas those injected within 5 or 10 min or more than 45 min after training did not. Avoidance was present until 24 h posttraining and absent after 48 h. Therefore, robust learning of the sickness-conditioned learning task occurs in one trial without the need for gustatory cues, and memory for the task lasts at least 24 h. Uses of this task to study memory formation in the day-old chick are discussed.

Overview of the International Conference on Particle Detection, Metrology, and Control.

The 1990 International Conference on Particle Detection, Metrology, and Control (February, 1990) jointly sponsored by the Parenteral Drug Association, Inc. and The Institute for Environmental Sciences provided a point in time to measure the progress achieved in the solution of the particle measurement problems that were defined at the 1987 Particle Conference. This Conference was of special significance since it addressed the scientific problems whose solution is required for successful harmonization of world particulate measurements and standards. A summary of the major papers presented follows.

Application of improved standardization methods and instrumentation in the USP particulate test for SVI.

The USP (788) requirement for particulate matter in small volume injections (SVI) became effective January 1, 1986. The standardization component of the requirement is time-consuming and open to subjective interpretation. Current generation light obscuration (LO) counters offer many advantages over those available when (788) was implemented. Significant improvements may be made to the requirement by optimizing the counter standardization procedure and making provisions for use of newer instrumentation. Key improvements to (788) suggested by our experience with the SVI test method include: (1) revision of the requirement to include a time-effective stand-alone standardization procedure; (2) provision for use of currently marketed LO counter systems; (3) development of new standard materials including a count standard; and, (4) deletion of the present requirement for validation of LO counts by microscopy. Significant user advantages accruing to an improved methodology and the use of new instrumentation will include decreased time spent in standardization, lower variability of data between different laboratories, and less instrument down time.

Method evaluation: automated microscopy as a compendial test for particulates in parenteral solutions.

Accurate enumeration and sizing of contaminant particles in parenteral solutions are critical to the assessment of product quality and suitability for patient use. Current manual microscopic and instrumental (light obscuration) methodologies specified by the USP both result in data of high variability. The microscopic assay for large volume parenterals is time consuming and incorporates an undesirable level of subjectivity; light obscuration counters tend to undersize larger particles and fibers and have low detection efficiency for some particle types commonly present in small volume injections. Light microscopic image analysis is proposed as a method which combines the best features of the two present methods and allows an accurate, precise, and cost effective analysis of parenteral contaminant particles. This paper briefly summarizes the principles of microscopic image analysis and discusses its application in concert with optimized sampling and counting techniques as an improved compendial methodology. Instrument performance requirements are discussed with reference to a number of currently available image analysis systems.

Effects of dietary 3-methylthiopropionate on metabolism, growth and hematopoiesis in the rat.

Rats were fed a basal diet supplemented with (2.57%) 3-methylthiopropionate (MTP) for 2 weeks. A marked depression in growth and food intake similar to that found in rats fed an equimolar level of methionine was observed. While supplemental glycine or serine alleviated the toxicity due to dietary methionine, similar levels added to the diets of rats fed MTP were without effect. The spleens of rats fed diets containing 2.57% MTP were grossly enlarged and darkened in comparison to spleens from control rats and histological examination of these spleens by light and transmission electron microscopy (TEM) revealed sequestration of large numbers of erythrocytes in the splenic sinusoids and red pulp similar to that seen in rats fed high levels of methionine. Marrow changes included increased numbers of erythroblastic islets and subtantial electron dense hemosiderin deposits in islet reticulum cells. Examination of peripheral blood erythrocytes by scanning electron microscopy revealed extensive variation in the size of the erythrocytes and the presence of large numbers of misshapen red cells in rats fed the diets containing MT. When viewed by TEM many erythrocytes had obvious membrane defects and remnants of cytoplasmic organellae. Many erythrocytes with reticulocyte morphology were present in the peripheral blood. This condition is characteristic of maturation arrest at the reticulocyte stage of development. The similarity of depression in growth and food intake and the identical abnormalities found in the spleens of rats fed high levels of MTP and methionine suggest that the transamination pathway of methionine catabolism may be important with respect to the toxicity of methionine. The ultrastructural changes noted in MTP-fed rats suggest a serious dysfunction of red cell hematopoiesis. The large numbers of defective and/or immature erythrocytes released from the marrow into the peripheral circulation, only to be later sequestered and destroyed in the spleen, is a reflection of a serious derangement.

Cytochemical characterization of cultured adult, guinea pig glomerular cells.

Methods are described for culture of intact or trypsin-digested adult guinea pig glomeruli. Cell types grown from intact glomeruli were distinctly different from those which dominated cultures of trypsin dissociated glomerular cells as determined by transmission and scanning electron microscopy as well as by fluorescence or enzyme cytochemical reaction with lectins and immunofluorescence cytochemical staining for actin or fibronectin. Cells with long cytoplasmic extensions (glomerular epithelial cells), cultured from intact glomeruli, have strong affinity for concanavalin-A and soybean agglutinin which react with glucose and galactose residues respectively. Rectangular cells (glomerular mesangial cells), cultured as the predominant cell from trypsinized glomeruli, have strong affinity for wheat germ agglutinin which reacts with N-acetyl glucosamine. Both of these cell types stained immunocytochemically for fibronectin and actin although the intracellular patterns were somewhat different. These two types of cells are able to secrete extracellular basement membrane material.

Pressure-flow relationships and pathological changes during renal preservation.

The renal pedicle of one kidney from each of four dogs was ligated for one hour. The contralateral kidney served as a control. Both kidneys were removed and perfused using the "Belzer" technique. Pressure-flow relationships were determined and biopsy samples taken. The vasculature was then injected with silicone rubber. Perfusion resistance, vascular filling with silicone rubber and observations made by electron microscopy were compared.

Enumeration and ultrastructure of C4-producing free alveolar cells from guinea pig lung.

Free alveolar cells from guinea pig lung producing the fourth emoponent of C (C4) were identified, enumerated, and characterized by using anti-C4 Fab-peroxidase conjugates in conjunction with transmission electron microscopy. The C4-producing cell population consisted of: 1) alveolar macrophages (AM); 2) less well differentiated phagocytes similar in morphology to exudate macrophages; and 3) weakly phagocytic secretory cells with numerous profiles of rough-surfaced endoplasmic reticulum (ER). Internal immunolabeling allowed the visualization of C4 in the ER, perinuclear space, and Golgi complex of producer cells and its release at cell surfaces; synthesis of C4 in vitro was sensitive to inhibitors both of protein synthesis and messenger RNA function. The percentage of free alveolar cells from normal animals competent for C4 production as indicated by cell surface immunolabeling was approximately 1% of the total cells obtained by lavage. Transnasal infection with Listeria monocytogenes, generation of a pulmonary granulomatous reaction by i.v. injection of heat-killed BCG, and aerosol infection of nonvaccinated animals with Myco-bacterium tuberculsois each resulted in an increase in numbers of AM and exudate macrophage-like free alveolar cells competent for C4-production.

Ultrastructure of effector--target cell interaction in secondary cell-mediated lympholysis.

The ultrastructure of the secondary cell-mediated lympholysis (CML) reaction and the effects on interacting lymphocytes of colchicine, cytochalasin B, and effector cell-specific antisera were examined using transmission and scanning electron microscopy. Surface labelling of cytotoxic secondary effector cells with cationized ferritin allowed them to be distinguished from unlabelled target lymphocytes. Effector--target interactions were characterized by intercellular junctions involving extensive areas of membrane apposition and interdigitation and extension of pseudopod-like processes by the effector cell. The abolition of such interactions when effector populations were pretreated with anti-Ly2 sera plus complement demonstrated target cell destruction in secondary CML to be dependent on the activity of restimulated cytotoxic T lymphocytes. Cytochalasin B and colchicine dramatically decreased the numbers of specific effector--target cell interactions observed. Although the data presented do not allow the possible activity of soluble lytic factors associated with the effector cell surface to be ruled out, they suggest that target cell lysis in the secondary CML system examined results from immune-specific binding of alloantigen-sensitized effectors to targets and osmotic effects which follow localized disruption of the target cell membrane.

Immune adherence in renal glomeruli. Complement receptor sites on glomerular capillary epithelial cells.

Several very recent reports have indicated the presence of receptor sites for the third component of complement in human but not other vertebrate renal glomeruli. The present study constitutes a demonstration that the glomerular capillary epithelial cell bears this receptor, detectable with either EAC complexes (EAC1423b) or fluores ceinated zymosan-C3 (ZC3b) complexes, Fresh, unfixed frozen sections of normal or diseased human kidneys, mechanically isolated human glomeruli, dissociated glomerular cells, and glomeruli and golmerular cells maintained in tissue culture were examined with various EAC complexes or ZC3b and examined by phase light microscopy, fluorescence microscopy, or transmission and scanning electron microscopy. Clearly, by scanning electron microscopy it was determined that glomerular capillary epithelial cells bind the immune-adherence EAC indicator cells. Because glomeruli or glomerular epithelial cells did not bind E, EA, EACI, EAC14, or EAC142 but did bind EAC1423b or ZC3b, it is concluded that C3b (activated bound fragment of the third component of complement) is responsible for the immune-adherence reaction in glomeruli. Preliminary examination of diseased renal biopsies indicates that sclerotic glomeruli, focal segmental sclerotic or proliferative glomerular capillary lesions, and proliferative epithelial crescents are immune-adherence negative. Furthermore, a clear or consistent inverse relationship between glomerular capillary deposits of C3 which presumably might block epithelial C3 receptor sites, and immune-adherence reactivity with EAC in vitro was not as evident in this study as reported previously by other investigators. Nevertheless, it is still attractive to conceive that glomerular C3 receptor sites might be responsible for binding of antigen-antibody-complement complexes and formation of immune-complex deposits, at least on the epimembranous (subepithelial) surface of glomerular capillary walls. Inability to demonstrate this immune-adherence phenomenon in glomeruli of other vertebrate animals suggests among other things that more investigation is necessary before ascribing a unique or universal significance to the C3 receptors identified in human glomeruli.