Non-patient related variables affecting levels of vascular endothelial growth factor in urine biospecimens

Vascular endothelial growth factor (VEGF) is an angiogenic protein proposed to be an important biomarker for the prediction of tumour growth and disease progression. Recent studies suggest that VEGF measurements in biospecimens, including urine, may have predictive value across a range of cancers. However, the reproducibility and reliability of urinary VEGF measurements have not been determined. We collected urine samples from patients receiving radiation treatment for glioblastoma multiforme (GBM) and examined the effects of five variables on measured VEGF levels using an ELISA assay. To quantify the factors affecting the precision of the assay, two variables were examined: the variation between ELISA kits with different lot numbers and the variation between different technicians. Three variables were tested for their effects on measured VEGF concentration: the time the specimen spent at room temperature prior to assay, the addition of protease inhibitors prior to specimen storage and the alteration of urinary pH. This study found that VEGF levels were consistent across three different ELISA kit lot numbers. However, significant variation was observed between results obtained by different technicians. VEGF concentrations were dependent on time at room temperature before measurement, with higher values observed 3-7 hrs after removal from the freezer. No significant difference was observed in VEGF levels with the addition of protease inhibitors, and alteration of urinary pH did not significantly affect VEGF measurements. In conclusion, this determination of the conditions necessary to reliably measure urinary VEGF levels will be useful for future studies related to protein biomarkers and disease progression.     

Heat shock protein 72 (Hsp72) specific induction and temporal stability in urine samples as a reliable biomarker of acute kidney injury (AKI)

Abstract

We demonstrated that urinary heat shock protein of 72 KDa (Hsp72) is a sensitive biomarker for the early detection of acute kidney injury (AKI). However, whether Hsp72 induction during an AKI episode is kidney-specific is unknown, as well as, the degree of Hsp72 stability in urine samples. In rats that underwent bilateral renal ischemia and reperfusion (I/R), Hsp72 levels were evaluated in several tissues and in collected urines under different storage and temperature conditions, as well as in variable numbers of freeze-thaw cycles. The effect of room temperature and five freeze-thaw cycles on urinary Hsp72 levels was also evaluated in urine samples from AKI patients. We found that Hsp72 increased exclusively in the renal cortex of I/R group, emphasizing its performance as an AKI biomarker. Urinary-Hsp72 remained constant at room temperature (48 h), during 9 months of storage and was not affected by five freeze/thaw cycles.     

Indices of immune function used by ecologists are mostly unaffected by repeated freeze-thaw cycles and methodological deviations

Background

Over the past couple of decades, measuring immunological parameters has become widespread in studies of ecology and evolution. A combination of different immunological indices is useful for quantifying different parts of the immune system and comprehensively assessing immune function. Running multiple immune assays usually requires samples to be repeatedly thawed and re-frozen. There is some evidence that repeated freezing and thawing can affect assay results, but this has never been comprehensively studied in some common ecological immunology assays. We tested the effect of multiple (1, 2, 3, 4, 5, 10) freeze-thaw cycles on the results of four commonly used immunological assays: haemolysis-haemagglutination titres, haptoglobin concentration, bacterial killing capacity and total immunoglobulins (IgY). We tested five different bird species from four different bird orders (Passeriformes, Columbiformes, Charadriiformes and Galliformes), and we included both captive and free-living individuals. In addition, we tested for haptoglobin concentrations and the haemolysis-haemagglutination assay if re-analysing samples 1 year apart led to different results. For the haemolysis-haemagglutination assay we also tested two different sources of rabbit blood, and we compared untreated microtitre plates with plates that were “blocked” to prevent nonspecific interactions between the plate and assay reagents.

Results

Repeated freezing and thawing of plasma had no effect on lysis titres, haptoglobin concentrations, bacterial killing capacity, or total immunoglobulin levels. Agglutination titres were unaffected by up to five cycles but were lower after ten freeze-thaw cycles. For the haemolysis-haemagglutination assay and haptoglobin concentrations, re-analysing samples 1 year apart yielded highly correlated data. For the haemolysis-haemagglutination assay, the source of rabbit blood did not influence the results, and the untreated vs. blocked plates differed slightly overall, but at the individual level assay results were highly correlated. Using different rabbit blood sources or different types of microtitre plates yielded highly correlated data.

Conclusions

Our data suggest that repeated freeze-thaw cycles do not impair assay results to the point of influencing ecological or evolutionary conclusions. Plasma samples can be safely stored in one tube and thawed repeatedly for different assays. Nevertheless, we recommend consistent treatment of samples in terms of freeze-thaw cycles or other laboratory treatments to minimize the potential for introducing a systematic bias.

     

Partition of free dolichol in human urine.

We have demonstrated that dolichol is present in the urinary supernatant. Most of the dolichol present in the supernatant seems to be associated with cellular debris or membrane fragments. The amount of sediment in healthy subjects correlate well with the volume of urine. Although it is illogical to express urinary dolichol relative to urine volume, a good correlation between the amount of sediment and urine volume has made its use justifiable. Because of the presence of a substantial amount of dolichol in the supernatant, it seems better to use uncentrifuged whole urine as the sample for measurement of dolichol.     

Cyclic cryopreservation affects the nanoscale material properties of trabecular bone

Tissues such as bone are often stored via freezing, or cryopreservation. During an experimental protocol, bone may be frozen and thawed a number of times. For whole bone, the mechanical properties (strength and modulus) do not significantly change throughout five freeze-thaw cycles. Material properties at the trabecular and lamellar scales are distinct from whole bone properties, thus the impact of freeze-thaw cycling at this scale is unknown. To address this, the effect of repeated freezing on viscoelastic material properties of trabecular bone was quantified via dynamic nanoindentation. Vertebrae from five cervine spines (1.5-year-old, male) were semi-randomly assigned, three-to-a-cycle, to 0–10 freeze-thaw cycles. After freeze-thaw cycling, the vertebrae were dissected, prepared and tested. ANOVA (factors cycle, frequency, and donor) on storage modulus, loss modulus, and loss tangent, were conducted. Results revealed significant changes between cycles for all material properties for most cycles, no significant difference across most of the dynamic range, and significant differences between some donors. Regression analysis showed a moderate positive correlation between cycles and material property for loss modulus and loss tangent, and weak negative correlation for storage modulus, all correlations were significant. These results indicate that not only is elasticity unpredictably altered, but also that damping and viscoelasticity tend to increase with additional freeze-thaw cycling.     

Photosynthetic response of the Antarctic moss Polytrichum alpestre Hoppe to low temperatures and freeze-thaw stress

The effect of low temperatures and freeze-thaw stress on photosynthetic carbon exchange in an Antarctic population of the turf-forming moss species Polytrichum alpestre Hoppe was investigated using infra-red gas analysis. Photosynthetic recovery from freezing was found to depend on the absolute depth of low temperature experienced. Repeated freeze-thaw cycles caused a greater reduction in gross photosynthesis than constant freezing over the same period of time suggesting that the freeze-thaw event itself, and not just cold temperatures, causes damage. The frequency of freeze-thaw events was significant: freeze-thaw cycles every 12 h inflicted more damage than freezethaw cycles every 24 or 48 h. Most damage occurred during the first cycle; relatively little was recorded during subsequent cycles. At +10°C, gross CO₂ flux was directly proportional to moss water content between 0.3 and 3.5 g·g^–1 dry mass. Moss samples with a low water content withstood freeze-thaw cycles to -5, -10 and-20°C better than samples with a high water content indicating that desiccation in the field may improve survival at low temperatures. Microclimate data for field populations of Polytrichum alpestre at Signy Island suggest that sub-zero temperatures and freeze-thaw stress may act as limiting factors on the species' distribution and viability, particularly when the insulating effect of snow cover is small.     

A Combined test using both cell sediment and supernatant cell‐free DNA in pleural effusion shows increased sensitivity in detecting activating EGFR mutation in lung cancer patients

Introduction

The aim of this study was to examine whether a combined test using both cell sediment and supernatant cytology cell‐free DNA (ccfDNA) is more useful in detecting EGFR mutation than using cell sediment DNA or supernatant ccfDNA alone in pleural effusion of lung cancer patients.

Methods

A total of 74 lung adenocarcinoma patients with paired samples between primary tumour and corresponding metastatic tumour with both cell sediment and supernatant ccfDNA of pleural effusion cytology were enrolled in this study. Cell sediment and supernatant ccfDNA were analysed separately for EGFR mutations by polymerase chain reaction.

Results

Out of 45 patients with mutant EGFR in primary tumours, EGFR mutations were detected in 23 cell sediments of corresponding metastases (sensitivity; 51.1%) and 20 supernatant ccfDNA corresponding metastases (sensitivity; 44.4%). By contrast, the combined test detected EGFR mutations in 27 corresponding metastases (sensitivity; 60.0%), and had a higher sensitivity than the cell sediment or the supernatant ccfDNA alone (P < .05). Out of 45 patients with mutant EGFR, 24, three and 18 were cytologically diagnosed as positive, atypical or negative, respectively. The detection rate in the combined test was highest (95.8%) in the positive group, and mutant EGFR was also detected in four of 18 samples (22.2%) in the negative group.

Conclusions

A combined test using both cell sediment DNA and supernatant ccfDNA samples increases the concordance rate of EGFR mutations between primary tumour and corresponding metastases. Our findings indicate that supernatant ccfDNA is useful even in cases where the cytological diagnosis is negative.     

Elevation of viral load by PCR and use of plasma preparation tubes for quantification of human immunodeficiency virus type 1

HIV-1 viral load testing from 51 patients was compared in plasma samples simultaneously processed and stored in primary Vacutainer plasma preparation tubes (PPTs) and PPT sample aliquots transferred in secondary tubes before freezing, using RT-PCR quantification with an ultrasensitive method (the Roche AMPLICOR HIV-1 MONITOR). We concluded that freezing the primary sample in the PPT collection tube can artificially elevate the HIV-1 viral load. We therefore store samples as frozen aliquots in a second tube.     

Impact of time between repeated sperm freezing cycles on sperm quality

Refreezing of sperm samples would provide the possibility of performing more cycles of fertility treatments. Although the effect of repeated cycles of freezing on sperm quality was studied, the effect of the length of the time interval between each freeze-thaw cycle has not been reported. Hence, we assessed the effect of incubation time on the sperm quality of thawed sperm after repeated freezing.One-hundred samples of potential sperm donations with normal sperm quality were evaluated. The fresh semen samples were analyzed and cryopreserved in liquid nitrogen until use. After thawing, the samples were divided randomly to two groups and reanalyzed for motility, vitality, and DNA fragmentation. They were incubated at room temperature and reanalyzed after either 90 min (group A) or 180 min (group B) of incubation, and once again after a repeated cycle of freezing and thawing.Our results showed that the sperm parameters of fresh samples of both groups were similar. After one freeze-thaw cycle, both groups still had comparable values. At the end of their respective incubation time periods, however, there was a significant difference in the mean values of the assessed parameters between the two groups (p < 0.01). An additional freeze-thaw cycle further exacerbated those differences, with group B undergoing an even more substantial decline (p < 0.001).Our data suggest that thawed human spermatozoa sustain a significant decline in sperm parameters in association with longer incubation time, which is further exacerbated by an additional freeze-thaw cycle.     

Effect of storage conditions of clinical materials on virological tests results by RT-PCR

Obtaining a reliable laboratory test result depends on many factors, among which preanalitical factors play a significant role. The aim of this study was to determine the effect of temperature, storage time of samples and number of freezing and thawing cycles on the results of tests carried out by RT-PCR. The study was conducted in a model of measles virus (RNA). The results revealed that: (1) Samples of clinical material (serum, cerebrospinal fluid, urine) for testing by RT-PCR can be stored for at least 5 days at room temperature or refrigerated and at least 7 days when frozen, (2) Freezing and thawing three times, samples of clinical material does not affect the outcome of the presence of viral RNA, (3) Isolated viral RNA is stable at freezer temperature and can be subjected to at least 20 freeze-thaw cycles without affecting the results.     

Soluble urokinase plasminogen activator receptor measurements: influence of sample handling

AIM: The influence of sample handling on soluble urokinase plasminogen activator receptor (suPAR) concentrations in serum and EDTA plasma was studied in 16 healthy premenopausal women. METHOD: Blood was collected in dry tubes and tubes containing EDTA and kept at 4 degrees C or 20 degrees C for 1, 3, 8, 24 or 72 hours before processing into serum or EDTA plasma. In addition, serum and EDTA plasma were frozen and thawed 1-8 times. All suPAR measurements were performed by ELISA. RESULTS: No significant differences were found between serum or EDTA plasma suPAR concentrations when whole blood samples were kept for 1, 3, 8 or 24 hours. Significantly higher suPAR levels were found in samples kept for 72 hours at 20 degrees C compared to samples processed into serum or EDTA plasma after short-term storage for no more than 24 hours after collection. No significant differences were observed when whole blood was kept at 4 degrees C for up to 72 hours. Repeated freezing and thawing had no significant effect on the serum and EDTA plasma suPAR levels. CONCLUSION: suPAR values in blood samples are dependent on the handling procedures of the samples. All samples of whole blood must be processed into EDTA plasma or serum within 24 hours if kept at 20 degrees C and within 72 hours if kept at 4 degrees C. However, repeated freezing/thawing cycles had no influence on suPAR values in the samples.     

Effects of freeze-thaw events on the viability of Cryptosporidium parvum oocysts in soil

The effects of freeze-thaw events on the inactivation of Cryptosporidium parvum oocysts in soil were examined. Oocysts were inoculated into distilled water in microcentrifuge tubes or into chambers containing soil the water content of which was maintained at 3%, 43%, or 78% of the container capacity. The chambers and tubes were then embedded in 3 soil samples from different aspects of a hillside landscape (Experiments 1 and 2) and in 3 distinct soil types (Experiment 3) and frozen at -10 C. Containers were thawed every 3 days for a period of 24 hr in 1-9 freeze-thaw cycles over 27 days (Experiments 1 and 2) and 2-5 freeze-thaw cycles over 15 days (Experiment 3). Oocyst viability was measured using the fluorescent dyes 4'6-diaminidino-2-phenylindole and propidium iodide. Inactivation rates were greater in soils than in water and greater in dry soil than in moist and wet soils. Soil type showed no effect on inactivation. Oocysts subjected to freeze-thaw cycles had inactivation rates not significantly different from those of oocysts subjected to -10 C under static conditions. The results indicated that 99% of oocysts exposed to soils that are frozen at -10 C will become inactivated within 50 days whether or not freeze-thaw cycles occur.     

Exercise lowers estrogen and progesterone levels in premenopausal women at high risk of breast cancer

Experimental and clinical data support a role for estrogens in the development and growth of breast cancer, and lowered estrogen exposure reduces breast cancer recurrence and new diagnoses in high-risk women. There is varied evidence that increased physical activity is associated with breast cancer risk reduction in both pre- and postmenopausal women, perhaps via lowered estrogen levels. The purpose of this study was to assess whether exercise intervention in premenopausal women at increased breast cancer risk reduces estrogen or progesterone levels. Seven healthy premenopausal women at high risk for breast cancer completed a seven-menstrual-cycle study. The study began with two preintervention cycles of baseline measurement of hormone levels via daily first-morning urine collection, allowing calculation of average area under the curve (AUC) hormone exposure across the menstrual cycle. Participants then began five cycles of exercise training to a maintenance level of 300 min per week at 80-85% of maximal aerobic capacity. During the last two exercise cycles, urinary estradiol and progesterone levels were again measured daily. Total estrogen exposure declined by 18.9% and total progesterone exposure by 23.7%. The declines were mostly due to decreased luteal phase levels, although menstrual cycle and luteal phase lengths were unchanged. The study demonstrated the feasibility of daily urine samples and AUC measurement to assess hormone exposure in experimental studies of the impact of interventions on ovarian hormones. The results suggest value in exercise interventions to reduce hormone levels in high-risk women with few side effects and the potential for incremental benefits to surgical or pharmacologic interventions.     

Specific gravity as an alternative to creatinine for estimating urine concentration in captive and wild chimpanzee (Pan troglodytes) Samples

The measurement of hormones in urine has become a widely used technique in primatology. Because urine concentration varies according to fluid intake, concentration must be measured in each sample collected, and hormone values are always expressed per unit of concentration. Traditionally, creatinine has been used as a concentration index, but some studies in humans have shown that creatinine varies among populations and even within and between individuals within a population, and that it begins to degrade after just one freeze-thaw cycle. In addition, creatinine measurement is relatively time-consuming and expensive and creates hazardous waste. In this study, we tested the hypothesis that specific gravity, or the ratio of the density of a sample to that of water, is highly correlated with creatinine measurement in urine samples collected from captive chimpanzees at the New Iberia Research Center in Louisiana and wild chimpanzees at the Ngogo study site in the Kibale National Park, Uganda. We found that specific gravity and creatinine were highly correlated in both captive (N=124) and wild (N=13) chimpanzee samples, and that specific gravity measurement was robust to actual and simulated transport conditions and repeated freeze-thaw cycles. We recommend that researchers consider specific gravity measurement as a preferable alternative to creatinine measurement in their studies of primate endocrinology.     

Freeze-thaw tolerance and clues to the winter survival of a soil community

Although efforts have been made to sample microorganisms from polar regions and to investigate a few of the properties that facilitate survival at freezing or subzero temperatures, soil communities that overwinter in areas exposed to alternate freezing and thawing caused by Foehn or Chinook winds have been largely overlooked. We designed and constructed a cryocycler to automatically subject soil cultures to alternating freeze-thaw cycles. After 48 freeze-thaw cycles, control Escherichia coli and Pseudomonas chlororaphis isolates were no longer viable. Mixed cultures derived from soil samples collected from a Chinook zone showed that the population complexity and viability were reduced after 48 cycles. However, when bacteria that were still viable after the freeze-thaw treatments were used to obtain selected cultures, these cultures proved to be >1,000-fold more freeze-thaw tolerant than the original consortium. Single-colony isolates obtained from survivors after an additional 48 freeze-thaw cycles were putatively identified by 16S RNA gene fragment sequencing. Five different genera were recognized, and one of the cultures, Chryseobacterium sp. strain C14, inhibited ice recrystallization, a property characteristic of antifreeze proteins that prevents the growth of large, potentially damaging ice crystals at temperatures close to the melting temperature. This strain was also notable since cell-free medium derived from cultures of it appeared to enhance the multiple freeze-thaw survival of another isolate, Enterococcus sp. strain C8. The results of this study and the development of a cryocycler should allow further investigations into the biochemical and soil community adaptations to the rigors of a Chinook environment.     

Chemical degradation of 3H-labeled substance P in tris buffer solution

Substance P (SP) is an important neuropeptide that has been implicated in several physiological processes, and it is necessary to devise an analytical procedure to measure endogenous SP with a combination of high sensitivity and maximum molecular specificity. However, the unique chemical nature of SP (polarity, chemical stability, ease of oxidation, peptide bond lability) plays a significant role in its analysis, such as in receptor assays, immunoassays, chromatography, and mass spectrometry. In this study, we evaluated in polypropylene and glass assay tubes the effects on the recovery and stability of tritiated SP ([3H]SP) of several pertinent experimental parameters such as buffer, pH, multiple freeze-thaw cycles, and incubation temperature and time. Bovine serum albumin (BSA) effectively reduced the absorption of [3H]SP to polypropylene and glass tube surfaces. Following multiple (6X) freeze-thaw cycles of solutions in BSA-precoated tubes, the recovery of radioactive [3H]SP remained high (greater than 75%) after the last cycle, whereas recovery was minimal in uncoated or siliconized glass tubes. A high level of radioactivity recovery was maintained for 14 days of storage of [3H]SP in triethylamine formate (TEAF) solution in BSA-precoated tubes at 4 and -20 degrees C, but decreased at 37 degrees C to less than 80% in only 3 h. Following storage in Tris-HCl (pH 7.4) buffer, a combination of HPLC and mass spectrometric analyses revealed that a significant amount of peptide bond cleavage occurred to produce the two peptides ArgProLys (RPK) and ArgProLysProGlnGln (RPKPQQ), with only a small amount of remaining intact SP. That decomposition was not observed in triethylamine formate TEAF (pH 3.14) buffer solutions.     

Effects of gamma irradiation and repetitive freeze-thaw cycles on the biomechanical properties of human flexor digitorum superficialis tendons

An increasing number of tissue banks have begun to focus on gamma irradiation and freeze-thaw in the reconstruction of anterior cruciate ligaments using allografts. The purpose of this study was to evaluate the biomechanical properties of human tendons after exposure to gamma radiation and repeated freeze-thaw cycles and to compare them with fresh specimens. Forty flexor digitorum superficialis tendons were surgically procured from five fresh cadavers and divided into four groups: fresh tendon, gamma irradiation, freeze-thaw and gamma irradiation+freeze-thaw. The dose of gamma irradiation was 25 kGy. Each freeze-thaw cycle consisted of freezing at -80 °C for 7 day and thawing at 25 °C for 6 h. These tendons underwent 4 freeze-thaw cycles. Biomechanical properties were analyzed during load-to-failure testing. The fresh tendons were found to be significantly different in ultimate load, stiffness and ultimate stress relative to the other three groups. The tendons of the gamma+freeze-thaw group showed a significant decrease in ultimate load, ultimate stress and stiffness compared with the other three groups. Gamma irradiation and repeated freezing-thawing (4 cycles) can change the biomechanical properties. However, no significant difference was found between these two processes on the effect of biomechanical properties. It is recommended that gamma irradiation (25 kGy) and repetitive freeze-thaw cycles (4 cycles) should not be adopted in the processing of the allograft tendons.     

Comparison of serum estradiol to urinary estrone conjugates in the rhesus macaque (Macaca mulatta)

Paired urine and serum samples were collected daily during fourteen nonconceptive (7 females) and ten conceptive (9 females) ovarian cycles from a total of 12 female rhesus monkeys (Macaca mulatta). Daily urine samples were analyzed for concentrations of estrone conjugates (Ei Conj). Serum samples were evaluated for concentrations of estradiol (E2) and progesterone (P) by radioimmunoassay (RIA), and bioactive luteinizing hormone (bLH) and monkey chorionic gonadotropin (mCG) were analyzed by a mouse Leydig cell bioassay. Linear correlation (r) between urinary E1 Conj and serum E2 (r range = -0.176-0.948) during nonconceptive cycles aligned by the preovulatory E1 Conj peak (Day 0) improved when daily hormone values were realigned to account for an approximately 24-h delay in the excretion of hormonal metabolites in urine (r range = 0.465-0.967). Similarly, correlation between urinary E1 Conj and serum E2 during conceptive cycles aligned by Day 0 (r range = 0.300-0.824) improved when values were offset by 24 h (r range = 0.408-0.876). When conceptive cycles were compared to nonconceptive cycles, serum P levels were significantly elevated over nonconceptive levels by Day +12 (p less than 0.001), and urinary E1 Conj levels by Day +13 (p less than 0.02), whereas serum E2 and bLH were both significantly elevated by Day +14 (p less than 0.0006 and p less than 0.01, respectively). In both nonconceptive and conceptive cycles, urinary E1 Conj paralleled serum E2 and demonstrated incremental increases above baseline levels, which were greater than for serum E2.     

Freeze-Thaw Tolerance and Clues to the Winter Survival of a Soil Community

Although efforts have been made to sample microorganisms from polar regions and to investigate a few of the properties that facilitate survival at freezing or subzero temperatures, soil communities that overwinter in areas exposed to alternate freezing and thawing caused by Foehn or Chinook winds have been largely overlooked. We designed and constructed a cryocycler to automatically subject soil cultures to alternating freeze-thaw cycles. After 48 freeze-thaw cycles, control Escherichia coli and Pseudomonas chlororaphis isolates were no longer viable. Mixed cultures derived from soil samples collected from a Chinook zone showed that the population complexity and viability were reduced after 48 cycles. However, when bacteria that were still viable after the freeze-thaw treatments were used to obtain selected cultures, these cultures proved to be >1,000-fold more freeze-thaw tolerant than the original consortium. Single-colony isolates obtained from survivors after an additional 48 freeze-thaw cycles were putatively identified by 16S RNA gene fragment sequencing. Five different genera were recognized, and one of the cultures, Chryseobacterium sp. strain C14, inhibited ice recrystallization, a property characteristic of antifreeze proteins that prevents the growth of large, potentially damaging ice crystals at temperatures close to the melting temperature. This strain was also notable since cell-free medium derived from cultures of it appeared to enhance the multiple freeze-thaw survival of another isolate, Enterococcus sp. strain C8. The results of this study and the development of a cryocycler should allow further investigations into the biochemical and soil community adaptations to the rigors of a Chinook environment.     

Repeated freeze–thaw cycles induce embolism in drought stressed conifers (Norway spruce,stone pine)

Freezing and thawing lead to xylem embolism when gas bubbles caused by ice formation expand during the thaw process. However, previous experimental studies indicated that conifers are resistant to freezing-induced embolism, unless xylem pressure becomes very negative during the freezing. In this study, we show that conifers experienced freezing-induced embolism when exposed to repeated freeze-thaw cycles and simultaneously to drought. Simulating conditions at the alpine timberline (128 days with freeze-thaw events and thawing rates of up to 9.5 K h(-1) in the xylem of exposed twigs during winter), young trees of Norway spruce [Picea abies (L.) Karst.] and stone pine (Pinus cembra L.) were exposed to 50 and 100 freeze-thaw cycles. This treatment caused a significant increase in embolism rates in drought-stressed samples. Upon 100 freeze-thaw cycles, vulnerability thresholds (50% loss of conductivity) were shifted 1.8 MPa (Norway spruce) and 0.8 MPa (stone pine) towards less negative water potentials. The results demonstrate that freeze-thaw cycles are a possible reason for winter-embolism in conifers observed in several field studies. Freezing-induced embolism may contribute to the altitudinal limits of conifers.