A new methodology to preserve the original proportion and integrity of cell-free fetal DNA in maternal plasma during sample processing and storage.

OBJECTIVE: To develop a standardized blood collection device that preserves fetal cell-free DNA and minimizes the cell-free DNA background in maternal plasma. METHODS: Blood samples were drawn from healthy pregnant donors into K(3)EDTA (BD vacutainer) and Cell-free DNA BCT tubes (Streck, Inc.) and kept at ambient temperature. Plasma was separated by centrifugation and cell-free DNA was extracted. Cell-free DNA from plasma was quantified by quantitative real-time polymerase chain reaction. RESULTS: Blood drawn into Cell-free DNA BCT tubes showed no change in the original proportion of fetal cell-free DNA during a 14-day storage period at ambient temperature. Conversely, maternal blood drawn into K(3)EDTA tubes showed a steady reduction in the original proportion of fetal cell-free DNA over the same time period. Using maternal plasma stored in Cell-free DNA BCT tubes for 14 days, fetal cell-free DNA was amplified 80-fold using whole genome amplification (WGA). CONCLUSION: Using Streck's Cell-free DNA BCT tubes, it is possible to preserve the original proportion of fetal cell-free DNA for extended times as well as minimize the post-sampling maternal cell-free DNA background. Preserved in this way, fetal cell-free DNA can be amplified by WGA technology to be used in prenatal diagnostic tests.

Electronic spectroscopy and photoisomerization of trans-urocanic acid in a supersonic jet.

trans-Urocanic acid (trans-UA), a component of the epidermal layer of skin, exhibits wavelength-dependent photochemistry. The quantum efficiency of isomerization to cis-UA is greatest when the molecule is excited on the long wavelength tail of its absorption profile in solution (300-320 nm). However, exciting the molecule where it absorbs UV light most efficiently (260-285 nm) causes almost no isomerization. We have used fluorescence excitation and dispersed emission methods in a supersonic jet to investigate the electronic states involved in this complex and interesting photochemistry. Three distinct regions are present in the excitation spectrum. Region I, which is below the isomerization barrier, contains sharp, well-resolved peaks that upon excitation emit from the S(1) state of trans-UA. Region II exhibits peaks that increase in broadness and decrease in intensity with increasing excitation energy. Upon excitation these peaks produce dual emission from the S(1) states of both trans- and cis-UA. The trans to cis isomerization barrier is estimated to be 1400 cm(-1). Region III exhibits excitation to the S(2) electronic state and has a broad structure that spans 3000 cm(-1) and occurs 4000 cm(-1) above S(1). S(2) excitation results in essentially no trans to cis isomerization.

Encapsulation of ribonucleic acid in human red blood cells for use as a reticulocyte quality control material for flow cytometric analysis.

The osmotic lysis procedure was employed to encapsulate ribonucleic acid (RNA) in human red blood cells in order to prepare a reticulocyte reference control. The procedure required the hypotonic dialysis of erythrocytes in the presence of RNA and cytosolic components of red blood cells followed by a short hypertonic dialysis to restore isotonicity and reseal the pores formed on the cell membrane during the hypotonic swelling. The procedure was monitored by a dedicated flow cytometer for reticulocyte counting and required 120 min. Approximately 20% of the erythrocytes undergoing the reversible osmotic lysis were encapsulated with various amounts of RNA. The morphology of the RNA-loaded erythrocytes were similar to those of normal erythrocytes and reticulocytes, however, their mean cell volume (MCV) was slightly smaller than normal cells. RNA-loaded erythrocytes prepared by this method were stable for several months as a reference control for identification and enumeration of reticulocytes using flow cytometric as well as manual analysis methods and resulted in a high correlation coefficient between these counting techniques.

A new platelet function test.

A method for screening EDTA(K3) blood samples for platelet function is described. The general availability of whole blood platelet counters in the clinical laboratory suggested their use to measure platelet aggregation. To simplify the collection and storage of blood samples, a method utilizing EDTA collected blood is described. Addition of calcium and citrate restores platelet function to blood samples even when stored at room temperature for several hours. Thus, the blood sample used to assay RBC, WBC and platelet parameters may also be employed in platelet aggregation. A comparison of this method with aggregometry on 120 subjects indicated a similar response to arachidonate, collagen, ristocetin and ADP.

Serum sperm agglutinating antibody formation in vasectomized men treated with dexamethasone.

The effect of immunosuppression with dexamethasone on sperm antibody formation following vasectomy in men was determined. Vasectomized men were treated with dexamethasone (4 mg) tablets starting two days prior to vasectomy and each day thereafter for a total of eight days for a total dose of 30 mg. There was no significant difference in serum sperm agglutinating antibody, cholesterol, triglycerides, or lipoproteins between the dexamethasone and placebo groups. The finding that immunosuppression at the time of vasectomy did not inhibit sperm antibody formation in man was surprising because in a similar study with Cynomoglus monkeys treatment at the time of vasectomy with dexamethasone inhibited sperm antibody development. The difference between the two studies may be that in monkeys sperm antigen presentation comes at the time of vasectomy and therefore immunosuppression at this time can prevent an immune response, but in man sperm antigen presentation comes at some later time following vasectomy. This species difference in sperm antigen presentation following vasectomy in man compared to monkeys may affect subsequent atherosclerosis development and explain why no cardiovascular disease is found in man following vasectomy.

Chemical induction of ovarian epithelial carcinoma in mice.

Murine ovaries were treated with silk sutures saturated with a solution of 7,12-dimethylbenz(a)anthracene in beeswax. One of 35 animals developed an epithelial carcinoma. This tumor was not successfully transplanted into young animals.

Sperm-agglutinating and -immobilizing antibody formation following vasectomy prevented with dexamethasone in cynomolgus monkeys.

Cynomolgus monkeys (Macaca fascicularis) were treated with 1.5 mg/kg dexamethasone (DEX) before (4 to 2 days) and after (0, 2, 4, and 7 days) vasectomy. Of the four monkeys treated with DEX, only one developed sperm antibody as measured by sperm-agglutinating and sperm-immobilizing assays. All six of the vasectomized monkeys not given DEX developed both agglutinating and immobilizing sperm antibodies. In this study, DEX given before and after vasectomy blocked sperm-agglutinating and -immobilizing antibody formation. We conclude that the major antigenic exposure to sperm responsible for sperm-agglutinating and -immobilizing antibody comes at the time of vasectomy.

Prevention of autoimmunization to spermatozoa by passive antibody.

The objective of this investigation was to determine if sperm antibody formation after vasectomy in guinea pigs can be inhibited by passive administration of antiserum to spermatozoa. Sperm antibody was obtained by bleeding vasectomized guinea pigs which had sperm-agglutinating antibody titers of 1 : 16 or higher. Gamma globulin was obtained by ammonium sulfate precipitation. Vasectomized guinea pigs were injected with immune gamma globulin and normal gamma globulin for a period of two weeks after vasectomy. In the group receiving normal gamma globulin the serum titer of sperm-agglutinating antibody reached 1 : 32 and remained at that level for duration of the study. In guinea pigs receiving immune gamma globulin detectable serum titers of sperm-agglutinating antibody did not develop. The investigation suggests that sperm antibody formation can be prevented by treating vasectomized animals with passive sperm antibody to spermatozoa.

Platelet adsorption-source of error in reference controls.

Preparation of platelet reference controls requires that the platelet membranes be strengthened by chemical cross-linking, usually with glutaraldehyde. The reaction produces a platelet which is strongly absorbed to plastic and glass surfaces. Tests of commercially available reference controls indicate adsorption to the counting containers. Surfactants inhibit adsorption but cause an apparent decrease in size. Polyethylene glyocol can eliminate adsorption without altering conductivity.

Initiation-promotion skin carcinogenesis: inhibition by cyclic and non-cyclic nucleotides.

The effect of nucleotides on initiation-promotion skin carcinogenesis in Swiss mice was investigated. Cyclic AMP was given before initiation with DMBA, between initiation and promotion, and at the same time as promotion with croton oil. Cyclic AMP was more effective in inhibiting tumor development when injected at the same as promotion with croton oil. 5'-adenosine-monophosphate (5'-AMP) and cyclic GMP were as effective as cyclic AMP in inhibiting tumor development under these conditions. However, adenosine, dibutyryl-cyclic AMP and 5'-guanosine-monophosphate (5'-GMP) were ineffective.

Stimulation of malignant skin cells by antibody to normal skin cells of mice.

The ability of antibodies developed against normal skin cells to stimulate skin cells transformed by 7,12-dimethylbenz[a]anthracene (DMBA) was investigated. Primary cultures of normal skin, containing both fibroblasts and epithelial cells, were established from epidermis of the back skin of adult strain A/J mice. Malignant skin cells were obtained by treating a subculture of normal cells with DMBA. Transformation was demonstrated by increased growth rate, growth in soft agar, and production of tumors in strain A/J mice. Antisera developed in New Zealand White rabbits against the normal cells were cytotoxic to both normal and malignant cells in the presence of complement of 1:320 dilution. However, greater dilutions of the antisera (1:500-1:1,000) in the absence of complement produced growth enhancement of the malignant but not of the normal cells. The growth-enhancing properties were present in the gamma-globulin fraction of the antisera that contained IgG, IgM, and IgA antibodies. Immunofluorescence studies indicated that antibodies from the sera bound to the membranes of both normal and malignant cells. These data indicate that antibodies to normal cells are stimulatory to DMBA-transformed cells and confirm previous data obtained with spontaneously transformed cells.

Antibody stimulation of benzo(a)pyrene carcinogenesis.

Benzo(a)pyrene (BP) was conjugated to horse serum albumin (HSA) and then attached to aldehyde fixed human erythrocytes. These cells were used in a passive hemagglutination test to measure BP antibody. BP antibodies were found to be induced in Swiss mice injected with tumorigenic doses of BP. Of the mice treated with BP, those which developed tumors soonest had the highest levels of BP antibody. This observation suggested that the antibody to BP may stimulate tumor development. When rabbit antibody to BP was injected with BP a significantly increased tumor formation occurred. Active immunization using BP conjugated to a foreign protein also significantly increased tumor formation when the mice were treated with BP. Our findings suggest that the immune response to carcinogens is an important component of the carcinogenic process.

Adenylate cyclase stimulation by trypsin.

Adenylate cyclase activity of a rat embryo fibroblast cell line (F111) is markedly increased by brief treatment with 1:300 trypsin. The degree of stimulation depends upon the length of time the cells are treated and the concentration of trypsin. Crystalline trypsin produced a stimulation similar to that obtained with 1:300 trypsin. Further, the addition of soybean trypsin inhibitor blocked the stimulation of adenylate cyclase by 1:300 trypsin. Trypsin-treated adenylate cyclase responds to PGE1, but there is no increase over that of untreated enzyme. This result and the increase in fluoride-stimulated levels of activity suggest that the trypsin is acting upon the catalytic unit of the enzyme.

Initiation-promotion skin carcinogenesis and immunological competence.

The immune competence of mice during initiation-promotion skin carcinogenesis was determined by skin allograft rejection and lymphocyte mitogenesis. The carcinogen 7, 12-dimethylbenzanthracene inhibited the cellular immune competence of mice while lymphocytes from croton oil treated mice had enhanced PWM response. Chlorphenesin, a stimulator of cellular immunity, was found to inhibit tumorigenesis in initiation-promotion skin carcinogenesis when injected during promotion.