Altered activity in cultured cells caused by contaminants in tubes widely used for blood collection and serum preparation

Summary Cell culture has been recognized as an extremely sensitive system for measuring the toxicity of various materials. A study was done to determine whether the type of tube used to collect blood or store human serum might affect results in experiments requiring blood drawn into such tubes. In order to test tubes for contaminants that might alter cellular activity, a variety of commercially available tubes used for collection of blood and storage of serum were shaken while containing culture medium with fetal bovine serum. The medium was then applied to 3T3 fibroblasts in culture. Measuring incorporation of tritiated thymidine into DNA in log phase cells as an index of cellular proliferation, it was found that medium containing serum preincubated in tubes routinely used for blood collection could be extremely toxic. The same types of tube were also used to prepare human serum. When serum from some of the tubes was applied to 3T3 fibroblasts, a stimulatory effect was observed, perhaps caused by selective adsorption of inhibitory components of the blood or serum by various tubes. It is, therefore, crucial in a properly controlled experiment using serum in vitro to collect blood in tubes that exert no toxic or stimulatory effects in the assay or, at least, to be consistent in one’s choice of tube. None of the tubes used for storage of serum showed significant effects in our assay.     

NO(x) contamination in laboratory ware and effect of countermeasures.

Contamination of various types of laboratory wares with NO(x) (NO(-)(2) and NO(-)(3)) was assessed systematically and the effect of extensive washing as a countermeasure was evaluated. Mean NO(x) contamination arising from a model procedure for NO(x) determination in plasma was 0.93 microM (range, 0.35-1.49 microM). The major source of contamination included conical tubes (54.8%) and pipette tips used for transfer of solution (12.3-16.3%). Except for soft glassware, most NO(x) contamination could be washed out by pure water. Although NO(x) contamination in respective laboratory wares could be reduced below detection levels by extensive washing, summation of the contamination through the model procedure could not be completely abolished (but the effect of washing persisted at least 10 days). Heavy contamination was noted in glassware (especially soft glass) and ultrafiltration units, which was difficult to remove. Several types of vacuum blood sampling tubes contained various levels of NO(x). Our results indicated that a small but significant amount of contamination remained in laboratory ware even after extensive washing, and that it is advisable to avoid the use of glassware (soft glass), ultrafiltration units, and vacuum blood sampling tubes during the processing of clinical sampling for the measurement of NO(x).     

Assessment of the contamination from devices used for sampling and storage of whole blood and serum for element analysis

An assessment of potential contamination risk associated with devices routinely used in hospitals and clinical laboratories for sampling and storage of whole blood and serum was made by analysis of leachates from the devices. The devices checked were disposable stainless steel needles, different types of blood collection tubes; serum separation tubes, disposable plastic pipettes and plastic vials used for serum storage. Concentrations of about 70 elements in solution after leaching with 0.05 mol l(-1) HNO3 were determined by double focusing sector field inductively coupled plasma mass spectrometry (sector field ICP-MS). For the elements present in blood/serum at concentrations higher than 10 ng ml(-1) (Na, Ca, Mg, P, Fe, Br, Si, Zn, Cu, Rb, Se and I) contribution from devices was as a rule negligible (less than 1% of expected concentrations in the body fluids), but for the majority of trace and ultra-trace elements it may significantly affect or even prevent accurate determination. The highest trace element contribution was found to derive from commercially available blood collection and serum separation tubes. Apparent concentrations of Al, Ba, Th, rare earth, and some other elements resulting from contamination were higher than normal serum concentrations all types of tubes tested for.     

Blood extraction from lancet wounds using vacuum combined with skin stretching.

Key factors and practical limits of blood extraction from lancet wounds on body sites other than the finger were determined by testing a large number of conditions. During these tests, the pain associated with lancing alternate body sites was rated as less painful than a fingerstick 98% of the time. Vacuum combined with skin stretching was effective in extracting an adequate volume of blood from the forearm for glucose testing, up to an average of 16 microl in 30 s. The amount of blood extracted increases with the application of heat or vacuum before lancing, the level of vacuum, the depth of lancing, the time of collection, and the amount of skin stretching. Vacuum and skin stretching led to significant increases, up to fivefold in the perfusion of blood in the skin as measured by laser Doppler. Our observations suggest that vacuum combined with skin stretching increases blood extraction at alternate sites by increasing the lancet wound opening, increasing the blood available for extraction by vasodilatation, and reducing the venous return of blood through capillaries.     

A Practical and Novel Method to Extract Genomic DNA from Blood Collection Kits for Plasma Protein Preservation

Laboratory tests can be done on the cellular or fluid portions of the blood. The use of different blood collection tubes determines the portion of the blood that can be analyzed (whole blood, plasma or serum). Laboratories involved in studying the genetic basis of human disorders rely on anticoagulated whole blood collected in EDTA-containing vacutainer as the source of DNA for genetic / genomic analysis. Because most clinical laboratories perform biochemical, serologic and viral testing as a first step in phenotypic outcome investigation, anticoagulated blood is also collected in heparin-containing tube (plasma tube). Therefore when DNA and plasma are needed for simultaneous and parallel analyses of both genomic and proteomic data, it is customary to collect blood in both EDTA and heparin tubes. If blood could be collected in a single tube and serve as a source for both plasma and DNA, that method would be considered an advancement to existing methods. The use of the compacted blood after plasma extraction represents an alternative source for genomic DNA, thus minimizing the amount of blood samples processed and reducing the number of samples required from each patient. This would ultimately save time and resources.The BD P100 blood collection system for plasma protein preservation were created as an improved method over previous plasma or serum collection tubes¹, to stabilize the protein content of blood, enabling better protein biomarker discovery and proteomics experimentation from human blood. The BD P100 tubes contain 15.8 ml of spray-dried K2EDTA and a lyophilized proprietary broad spectrum cocktail of protease inhibitors to prevent coagulation and stabilize the plasma proteins. They also include a mechanical separator, which provides a physical barrier between plasma and cell pellets after centrifugation. Few methods have been devised to extract DNA from clotted blood samples collected in old plasma tubes^2-4. Challenges from these methods were mainly associated with the type of separator inside the tubes (gel separator) and included difficulty in recovering the clotted blood, the inconvenience of fragmenting or dispersing the clot, and obstruction of the clot extraction by the separation gel.We present the first method that extracts and purifies genomic DNA from blood drawn in the new BD P100 tubes. We compare the quality of the DNA sample from P100 tubes to that from EDTA tubes. Our approach is simple and efficient. It involves four major steps as follows: 1) the use of a plasma BD P100 (BD Diagnostics, Sparks, MD, USA) tube with mechanical separator for blood collection, 2) the removal of the mechanical separator using a combination of sucrose and a sterile paperclip metallic hook, 3) the separation of the buffy coat layer containing the white cells and 4) the isolation of the genomic DNA from the buffy coat using a regular commercial DNA extraction kit or a similar standard protocol.     

The effect of pre-analytical variability on the measurement of MRM-MS-based mid- to high-abundance plasma protein biomarkers and a panel of cytokines

Blood sample processing and handling can have a significant impact on the stability and levels of proteins measured in biomarker studies. Such pre-analytical variability needs to be well understood in the context of the different proteomics platforms available for biomarker discovery and validation. In the present study we evaluated different types of blood collection tubes including the BD P100 tube containing protease inhibitors as well as CTAD tubes, which prevent platelet activation. We studied the effect of different processing protocols as well as delays in tube processing on the levels of 55 mid and high abundance plasma proteins using novel multiple-reaction monitoring-mass spectrometry (MRM-MS) assays as well as 27 low abundance cytokines using a commercially available multiplexed bead-based immunoassay. The use of P100 tubes containing protease inhibitors only conferred proteolytic protection for 4 cytokines and only one MRM-MS-measured peptide. Mid and high abundance proteins measured by MRM are highly stable in plasma left unprocessed for up to six hours although platelet activation can also impact the levels of these proteins. The levels of cytokines were elevated when tubes were centrifuged at cold temperature, while low levels were detected when samples were collected in CTAD tubes. Delays in centrifugation also had an impact on the levels of cytokines measured depending on the type of collection tube used. Our findings can help in the development of guidelines for blood collection and processing for proteomic biomarker studies.     

Untersuchungen über das Pollenschlauchwachstum von Pisum sativum und einiger mutanten

Summary When the pollen tubes of Pisum sativum (initial line) and of its mutants are grown on a standard medium containing only sucrose, boric acid and agar-agar, no difference in maximum length was observed. But, while pollen tubes of the initial line took nine hours to reach this length, pollen tubes of the mutants needed only six hours. Growth seems to be faster in pollen tubes of the mutants than in those of the initial line.Further investigations examined the influence of twenty-one amino acids on pollen tube growth. With the initial line, these substances can be classified into three groups: those that promote pollen tube growth; those which have no influence upon its growth; and those which reduce its growth. The amino acids of each group are characterized by special structural properties. Those amino acids which accelerate pollen tube growth of the initial line show variable effects on the pollen tubes of the mutants. In some cases the same behaviour of pollen tubes can be observed whether amino acids are added or not, in others the addition of amino acids has a positive effect on pollen tube growth, though less than on pollen tubes of the initial line, and in a single case the addition of an amino acid is followed by a negative effect on growth.     

Evaluation of blood collection tubes using selected reaction monitoring MS: Implications for proteomic biomarker studies

An emphasis of current proteomic research is the validation of plasma protein biomarkers. The process of blood collection itself is critical to the accuracy and reproducibility of quantitative biomarker assays. We have developed selected reaction monitoring (SRM) assays to analyse thirteen abundant plasma proteins and evaluated the impact of three different blood collection tubes on the levels of these proteins. We also assessed the implications of the time taken to analyse plasma samples by evaluating the recovery of these proteins. We showed that SRM detects minor differences in the levels of some proteins which can be attributed to collection tube type. The average recovery for 12 of 18 assays was higher for proteins that were collected in tubes containing protease inhibitors compared to conventional collection tubes. For five of the assays, the differential recovery was statistically significant. Delaying MS analysis of a freeze‐thawed sample for 1 hour showed greatly reduced recovery of these analytes; however differences attributed to tube type were only evident at the baseline timepoint. Finally, we assessed the natural variation of circulating levels of these proteins in a cohort of seven healthy individuals. This study provides useful information for researchers contemplating blood collection for undertaking protein biomarker studies.     

Measurements of oxygen production rate in flowing Spirulina suspension

Summary The oxygen production rates for a cyanobacterial suspension flowing in straight and coiled tubes were measured to find a way of achieving higher efficiency of light utilization by means of convective mixing. The photosynthetic flow chambers were made of glass tubes and illumination was by fluorescent light. The cyanobacterium used was Spirulina platensis, which has a high growth rate. The oxygen production rate for fluid flow in straight and coiled tubes increase with the increase in Reynolds number. The maximum oxygen production rate was achieved at 30°C for both tube reactors, but the oxygen production rate was higher for the coiled tube unit than the straight tube unit at 30°C. Thus the convective mixing generated in the coiled tube reactor contributed to an increased in light utilization, which played an important part in improving the oxygen production rate. Offprint requests to: K. Tanishita     

Use of silica gel polymer for DNA extraction with organic solvents

Phenol and chloroform are the standard solvents used for DNA extraction. These solvents aid in the removal of protein and lipid from crude or partially purified cell extracts. Although the procedure is well established, the solvents are noxious, caustic, and unpleasant. We describe in this paper the use of a special blood collection tube to isolate the offensive organic solvents. With the use of silica gel polymer containing tubes, phenol, phenol:chloroform, or chloroform can be separated from the DNA containing aqueous phase in a rapid and safe manner. The method permits higher yields of DNA since the DNA is poured from the tube rather than aspirated with pipet.     

Evaluation of micro serum separator tubes for obtaining serum specimens suitable for serologic testing

Paired blood samples from Sprague Dawley rats were collected in micro serum separator tubes and in conventional 2 ml volume blood collection tubes. Commercially available ELISA test kits for Mycoplasma pulmonis and Sendai virus were used to compare the two collection systems. No significant differences were found in the optical density values between the two collection systems.     

A freeze-capture method for the study of platelet-sized particle distributions

A rapid freezing method was developed to study the distributions of fluorescent platelet-sized particles in flows of blood suspensions through thin-walled capillary tubes. Segments of frozen tubes were mounted in a refrigerated microtome on the stage of an epifluorescence microscope. Sections of tube were cut away, images of newly exposed cross-sections were recorded on video tape, and distances of the particles from the wall were measured from recorded images. The distance data were used to construct histograms that were proportional to the local concentration. Results indicated that this method is suitable for the study of the distribution of platelet-sized particles over a wide range of hematocrit, that the basic profile is reproducible to within 15%, and that the non-uniform profile is not a result of events at the tube entrance.     

负压封闭引流技术在躯干部皮肤恶性肿瘤切除术中的应用

目的:探讨负压封闭引流技术应用于躯干部皮肤恶性肿瘤切除术的临床效果,并分析其临床应用价值。方法:回顾性分析2012年5月~2015年3月我院收治的11例躯干部皮肤恶性肿瘤患者的临床资料,均予行肿物切除术并同时行自体皮游离移植术,并外用持续负压封闭引流术于植皮术区。结果:所有患者均接受肿物切除+植皮+封闭负压引流技术治疗。9例患者于4~9天后拆除负压材料,皮片全部成活。1例患者治疗后24小时内吸引管堵塞,经术后于24小时内行管路冲洗疏通后再次行负压吸引,于术后5天首次拆除负压材料,见皮片部分成活,部分皮片仍有浮动,血运未明显建立,予以扩大皮片引流孔后再次行负压封闭引流术,术后9天再次拆除负压材料,见皮片成活较好。1例患者治疗4天后出现新鲜渗血经引流管引出,予拆除负压材料,所植皮片下有积血块,有新鲜渗血,予以清创后再次行负压封闭引流术,术后9天再次拆除负压材料,见皮片成活较好。随访半年到3年,所植皮片无破溃,肿瘤无复发。结论:躯干部皮肤恶性肿瘤切除术中,植皮联合封闭式负压引流技术可使所植皮片固定确实,充分引流渗液积血,利于皮片成活,对无法有效包扎固定肢体特殊部位(肩部、臀部、躯干部)等术区植皮提供了有效方法,具有一定的推广应用价值。     

Improved rapid sampling for in vivo kinetics of intracellular metabolites in Saccharomyces cerevisiae.

An integrated approach is used to develop a rapid sampling strategy for the quantitative analysis of in vivo kinetic behavior based on measured concentrations of intracellular metabolites in Saccharomyces cerevisiae. Emphasis is laid on small sample sizes during sampling and analysis. Subsecond residence times are accomplished by minimizing the dead volume of the sterile sampling system and by maximizing flow rates through application of vacuum to the sampling tubes in addition to the overpressure in the fermenter. A specially designed sample tube adapter facilitates sampling intervals of 4 to 5 s for various test tube types. Statistical analysis of the results obtained from enzymatic and liquid chromatography mass spectrometry (LC-MSMS) analysis of the metabolite concentrations was used to optimize the sampling protocol. The most notable improvement is reached through the introduction of vacuum drying of the cell extract. The presented system is capable of reliably dealing with fermenter samples as small as 1-g with a variation of less than 3%, and is thus ideally suited for intracellular measurements on small, lab-scale fermenters.     

Blood Substrate Collection and Handling Procedures under Pseudo-Field Conditions: Evaluation of Suitability for Inflammatory Biomarker Measurement

Routine incorporation of blood-based biomarker measurements in population studies has been hampered by challenges in obtaining samples suitable for biomarker assessment outside of laboratory settings. Here, we assessed the suitability of venous blood left unprocessed for 4, 24, or 48 hours post-collection at either room temperature or 4°C for quantification of two biomarkers, Interleukin-6 (IL-6) and C-reactive protein (CRP). Blood samples were collected in both K₂EDTA tubes and a dedicated plasma-preservation tube, P100. Dried blood spot (DBS) samples from the same subjects were also collected in order to compare delayed-processing plasma performance against a popular alternative collection method. We found that K₂EDTA mean plasma concentrations of both IL-6 and CRP were not significantly different from concentrations in plasma processed immediately; this was observed for tubes stored up to 48 hours pre-processing at either temperature. Concentrations of IL-6 measured in P100 tubes showed significant time-dependent increases when stored at room temperature; otherwise, levels of IL-6 and CRP were similar to those found in samples processed immediately. Levels of CRP in DBS were correlated with plasma CRP levels, even when pre-processed blood was stored for up to 48 hours. These data indicate that plasma is suitable for IL-6 and CRP estimation under data collection conditions that involve processing delays.     

Blood collection from the facial (maxillary)/musculo-cutaneous vein in true frogs (family Ranidae)

Collection of blood from amphibians, as in other classes of vertebrate animals, is essential to evaluate parameters of health, diagnose hemoparasitism, identify viral and bacterial pathogens, and measure antibodies. Various methods of blood collection have been described for amphibians. Most can be cumbersome (venipucture of femoral vein, ventral abdominal vein or lingual venus plexus) or result in pain or deleterious health consequences (cardiac puncture and toe-clipping). We describe an easy and practical technique to collect blood from frogs and toads that can be used in multiple species and is minimally invasive. The technique consists of puncturing either the facial or, less commonly, the musculo-cutaneous vein and collecting the blood with a capillary tube. These veins run dorsal and parallel to the maxillary bone and can be accessed by quick insertion and withdrawal of a needle through the skin between the upper jawline and the rostral or caudal side of the tympanum. The needle should be of 27 or 30 gauge for anurans weighing more or less than 25 g, respectively. Although the technique has been used by some amphibian researchers for years, it is little known by others and has never been fully described in a peer-reviewed publication.     

Biomarkers for Monitoring Pre-Analytical Quality Variation of mRNA in Blood Samples

There is an increasing need for proper quality control tools in the pre-analytical phase of the molecular diagnostic workflow. The aim of the present study was to identify biomarkers for monitoring pre-analytical mRNA quality variations in two different types of blood collection tubes, K₂EDTA (EDTA) tubes and PAXgene Blood RNA Tubes (PAXgene tubes). These tubes are extensively used both in the diagnostic setting as well as for research biobank samples. Blood specimens collected in the two different blood collection tubes were stored for varying times at different temperatures, and microarray analysis was performed on resultant extracted RNA. A large set of potential mRNA quality biomarkers for monitoring post-phlebotomy gene expression changes and mRNA degradation in blood was identified. qPCR assays for the potential biomarkers and a set of relevant reference genes were generated and used to pre-validate a sub-set of the selected biomarkers. The assay precision of the potential qPCR based biomarkers was determined, and a final validation of the selected quality biomarkers using the developed qPCR assays and blood samples from 60 healthy additional subjects was performed. In total, four mRNA quality biomarkers (USP32, LMNA, FOSB, TNRFSF10C) were successfully validated. We suggest here the use of these blood mRNA quality biomarkers for validating an experimental pre-analytical workflow. These biomarkers were further evaluated in the 2^nd ring trial of the SPIDIA-RNA Program which demonstrated that these biomarkers can be used as quality control tools for mRNA analyses from blood samples.     

Blood cells in rainbow trout Oncorhynchus mykiss milt: relation to milt collection method and sampling period

The presence of blood cells in milt of rainbow trout (Oncorhynchus mykiss) collected every week between the middle at the end of the spawning season, either by stripping or by catheterization was investigated. Basic sperm biological and biochemical characteristics were also evaluated. Because milt often becomes contaminated with blood during collection, we also studied the influence of experimental blood contamination on sperm motility and biochemical parameters of seminal plasma. We demonstrated the presence of blood cells (erythrocytes, lymphoid, and phagocytes) in rainbow trout milt collected by both methods. Both sampling period and collection method influenced sperm characteristics, however the relationship between these characteristics and blood cells are not clear at present. A high number of blood cells in milt was found in some samples, possibly due to inflammation, because at the same time we observed bacteria and elevated levels of protein and antiproteinase activity in contaminated samples. Experimental contamination of milt with blood did not influence sperm motility, protein concentration and LDH activity of the 5-day-stored semen. Our study demonstrated that blood cells were present in rainbow trout milt. Blood cells may also appear in milt as a result of bleeding and their elevated levels are present during inflammation.     

A semi-empirical model for flow of blood and other particulate suspensions through narrow tubes

A semi-empirical model applicable to the flow of blood and other particulate suspensions through narrow tubes has been developed. It envisages a central core of blood surrounded by a wall layer of reduced hematocrit. With the help of this model the wall layer thickness and extent of plug flow may be calculated using pressure drop, flow rate and hematocrit reduction data. It has been found from the available data in the literature that for a given sample of blood the extent of plug flow increases with decreasing tube diameter. Also for a flow through a given tube it increases with hematocrit. The wall layer thickness is found to decrease with increase in blood hematocrit. A comparison between the results of rigid particulate suspensions and blood reveals that the thicker wall layer and smaller plug flow radius in the case of blood may be attributed to the deformability of the erythrocytes.