Storage of blood for methemoglobin determination: comparison of storage with a cryoprotectant at -30 degrees C and without any additions at -80 degrees C or -196 degrees C

Changes in methemoglobin (Met-Hb) concentrations during storage of whole blood or mixtures of blood and a cryoprotectant at refrigerated or various freezing temperatures were examined using blood samples from nitrite-administered rats and from autopsy cadavers. When whole blood was stored at 3 degrees C, Met-Hb reduction was observed in blood samples from nitrite-administered rats and in the blood from a victim poisoned by a weed killer containing some oxidant. When samples were stored at -30 degrees C, Met-Hb formation by autoxidation was inevitably observed in blood samples stored as whole blood, whereas addition of a cryoprotectant to whole blood could prevent Met-Hb formation in all the blood samples. When whole blood was stored at -80 degrees C or -196 degrees C, Met-Hb concentrations were practically stable until at least 30 days regardless of the initial values except in the control rat blood samples stored at -80 degrees C which showed slight formation of Met-Hb. From the results obtained, both the storage with a cryoprotectant at -30 degrees C and that without any additions at -80 degrees C or -196 degrees C proved to be suitable for long-term storage of blood samples from autopsy cadavers for Met-Hb determination.     

The effect of storage at various temperatures on blood alcohol concentration

There is a paucity of data available on the effect of storage on blood alcohol concentration (BAC) at elevated temperatures. Changes in serum alcohol concentration (SAC) and BAC were studied. Serum samples spiked with alcohol in the presence or absence of preservative were stored at 26.7 °, 32.2 ° or 37.8 °C respectively. Serum alcohol concentrations were determined daily on days 1 through 14, and on days 21 and 35. Under these controlled conditions, no significant change in SAC was observed at the aforementioned temperatures. Whole blood samples submitted from outside agencies were initially analyzed (day 1), then stored for 35 days at different elevated temperatures before a second analysis. The average loss in BAC was 19.20 ± 15.6, 9.95 ± 5.7, and 15.60 ± 6.9% when the samples were stored at 26.7, 32.2 and 37.8 °C, respectively. The alcohol loss from whole blood samples may be attributed to chemical oxidation rather than to elevated temperatures. It is, therefore, concluded that a whole blood sample obtained from a living individual and stored in a locker, glove compartment or other environment where the temperature is elevated, may lose 10–19% of its alcohol content over 35 days of storage. On the other hand, when a serum or plasma sample is exposed to the same environment, no significant change in SAC was observed. The utility of this information is significant to the forensic toxicologist. The results of this study suggest that a whole blood sample analyzed after exposure to elevated temperature may have had, originally, a higher BAC.     

In vitro production of GHB in blood and serum samples under various storage conditions

The in vitro production of GHB was observed in freshly collected, untreated whole blood samples using glass BD-Vacutainers and polypropylene S-monovettes. GHB concentrations were determined daily over a period of one week and after 3, 6 and 9 weeks again. Furthermore, the GHB concentration in 40 untreated random whole blood samples stored at 4°C for a longer period of time (10 samples 12 month, 10 samples 24 month and 20 samples 36 month) was also determined. For comparison, the in vitro production of GHB in freshly collected and prepared serum samples was observed. GHB serum concentrations were determined three times over a period of one week and once again after six weeks. Sample preparation was performed by means of methanolic extraction following the precipitation of whole blood and serum samples. A methanolic standard calibration was done in a low range of 0.005-0.1 μg/mL (LOD: 0.004, LLOQ: 0.013). For quantification a spiked blood bank serum with a determined GHB concentration of 0.09 μg/mL was used. Corrected calibrations in the range of 0.09-5.09 μg/mL were used (LOD: 0.08 μg/mL, LLOQ: 0.30 μg/mL), recovery: 91.3% (high level: 4.09 μg/mL) 50.5% (low level: 0.19 μg/mL). RESULTS: Relevant elevation of GHB was observed in all whole blood samples stored in liquid form (4°C or room temperature). In two of the 40 whole blood samples stored over a longer period of time at 4°C, GHB concentrations in the range of 13 μg/mL were even determined. These findings constitute grounds for caution. Even a GHB cut-off level of 5 μg/mL cannot be considered as "absolutely positive" proof of a case of exogenous administration, at least in untreated liquid blood samples in long time storage. However, no significant elevations of GHB were otherwise observed in any of the serum samples independently of storage temperature nor in the whole blood samples that were frozen for storage. CONCLUSIONS: The results suggest that the cut-off for exogenous GHB of 5 μg/mL could be lowered significantly, with the consequence of winning valuable time for the potential victim, but only if serum is collected for GHB determination or if the whole blood sample is frozen immediately after collection and the procedure well documented.     

Stability of blood carbon monoxide and hemoglobins during heating

The effects of heating on hemoglobin (Hb) and carbon monoxide (CO) levels in human blood were investigated by in vitro experiments. Head-space gas chromatography (HS-GC) using a molecular sieve 5A stationary phase and thermal conductivity detection was adopted for the measurement of CO gas, and spectrophotometric methods were used for the measurement of various Hb forms, protein and heme contents. Deteriorated absorbance spectra were observed for heat-treated blood samples, and double wavelength spectrophotometry was proven to give wrong percent saturation of carboxyhemoglobin content (% CO-Hb). The blood sample taken from one fatal fire casualty gave significantly higher % CO-Hb measured spectrophotometrically, compared to that by HS-GC. Control blood or purified Hb solution, which was saturated with CO in designated extent, was heated in a sealed vial. Under the incubation below 54 degrees C, all Hb forms were stable, except for oxyhemoglobin (Hb-O(2)), which was partially oxidized to met-hemoglobin (Met-Hb). In contrast, under the incubation at 65 degrees C, Met-Hb was denatured completely to be insoluble, and Hb-O(2) was partially denatured via Met-Hb formation. CO-Hb was resistant against heating. The difference of heat susceptibility and precipitability among Hb forms resulted in artificial increase of % CO-Hb. During heating, spontaneous CO was produced from blood.     

Stability study of the designer drugs "MDA, MDMA and MDEA" in water, serum, whole blood, and urine under various storage temperatures.

A controlled study was undertaken to determine the stability of the designer drugs MDA, MDMA and MDEA in pooled serum, whole blood, water and urine samples over a period of 21 weeks. The concentrations of the individual designer drugs in the various matrices were monitored over time, in the dark at various temperatures (-20, 4 or 20 degrees C), for a low (+/- 6 ng/ml for water, serum and whole blood and +/- 150 ng/ml for urine) and a high concentration level (+/- 550 ng/ml for water, serum and whole blood and +/- 2500 ng/ml for urine). Compound concentrations were measured using a validated HPLC assay with fluorescence detection. Our study demonstrated no significant loss of the designer drugs in water and urine at any of the investigated temperatures for 21 weeks. The same results were observed in serum for up to 17 weeks, and up to 5 weeks in whole blood. After that time, the compounds could no longer be analyzed due to matrix degradation, especially in the low concentration samples that were stored at room temperature. This study demonstrates that the designer drugs, MDA, MDMA and MDEA are stable when stored at -20 degrees C for 21 weeks, even in haemolysed whole blood.     

Gamma-hydroxybutyric acid stability and formation in blood and urine

Gamma-hydroxybutyric acid (GHB) can cause problems in interpretation of toxicological findings due to its endogenous nature, significant production in tissues after death and potential formation in stored samples. Our study was designed to determine the influence of storage conditions on GHB levels and its possible in vitro formation in blood and urine in cases where no exogenous use of GHB or its precursors was suspected. The samples were prepared by validated method based on liquid-liquid reextraction with adipic acid internal standard and MSTFA derivatization and assayed on a GC-MS operating in EI SIM mode. The first part of the study was performed with pooled blood and urine samples obtained from living and deceased subjects stored with and without NaF (1% w/v) at 4 and -20 degrees C over 8 months. In ante-mortem samples (both blood and urine) no significant GHB production was found. After 4 months of storage, the substantial GHB rise up to 100 mg/Lwas observed in post-mortem blood stored at 4 degrees C without NaF with subsequent gradual decrease in following months. The inhibition of GHB production was apparent during storage in NaF treated frozen blood samples. In post-mortem urine only slight temporary GHB levels were ascertained (up to 8 mg/L). The second part of our study was aimed to analyse 20 individual post-mortem blood samples stored at 4 degrees C for 16-27 days between autopsy and analysis without preservation followed by storage at 4 degrees C with NaF for 4 months. The temporary GHB production with maximum of 28 mg/Lwas detected in some samples.     

Headspace gas chromatographic determination of ethanol: the use of factorial design to study effects of blood storage and headspace conditions on ethanol stability and acetaldehyde formation in whole blood and plasma

Our headspace gas chromatographic flame ionization detection (HS-GC-FID) method for ethanol determination showed slightly, but consistently, low ethanol concentrations in whole blood (blood) in proficiency testing programs (QC-samples). Ethanol and acetaldehyde were determined using HS-GC-FID with capillary columns, headspace equilibration temperature (HS-T degrees ) of 70 degrees C and 20 min equilibration time (HS-EqT). Full factorial designs were used to study the variables HS-T degrees (50 degrees -70 degrees C), HS-EqT (15-25 min), ethanol concentration (0.20-1.20 g/kg) and storage at room temperature (0-6 days) with three sample-sets; plasma, hemolyzed blood and non-hemolyzed blood. A decrease in the ethanol concentration in blood was seen as a nearly equivalent increase in the acetaldehyde concentration. This effect was not observed in plasma, indicating chemical oxidation of ethanol to acetaldehyde in the presence of red blood cells. The variables showed different magnitude of effects in hemolyzed and non-hemolyzed blood. A decrease in ethanol concentration was seen even after a few days of storage and also when changing the HS-T degrees from 50 to 70 degrees C. The formation of acetaldehyde was dependent on all the variables and combinations of these (interactions) and HS-T degrees was involved in all the significant interaction effects. Favorable instrumental conditions were found to be HS-T degrees of 50 degrees C and HS-EqT of 15-25 min. The ethanol concentrations obtained for the range 0.04-2.5 g/kg after analyzing authentic forensic blood samples with a HS-T degrees of 50 degrees C were statistically significantly higher than at 70 degrees C (+0.0154 g/kg, p < 0.0001, n = 180). In conclusion, chemical oxidation of ethanol to acetaldehyde in the presence of red blood cells has been shown to contribute to lowered ethanol concentrations in blood samples. Storage conditions before analysis and the headspace equilibration temperature during analysis were important for the determination of blood ethanol concentrations.     

Instability of pancuronium in postmortem blood and liver taken after a fatal intramuscular Pavulon injection

The present study was designed to determine the stability of pancuronium in postmortem blood and liver during storage. Results were obtained using the method by Kerskes et al. [C.H.M. Kerskes, K.J. Lusthof, P.G.M. Zweipfenning, J.P. Franke, The detection and identification of quaternary nitrogen muscle relaxants in biological fluids and tissues by ion-trap LC-ESI-MS, J. Anal. Toxicol. 26 (2002) 29-34.], modified and validated in our laboratory. Target analytes were isolated after enzymatic hydrolysis followed by solid phase extraction (BondElut C18 column). Internal standardisation was carried out using laudanosine and the target ions were monitored by LC-ESI-MS (monitoring ions m/z 358 for IS and 286 for pancuronium). Materials were taken from a 46-year-old woman, who had been found dead. A syringe (2 ml) and an empty ampoule of Pavulon (4 mg/2 mL) were found in her hand. The residual volume of fluid in the syringe was 0.7 ml. An autopsy was performed six days after death. It revealed a needle mark on the left thigh. Postmortem materials (muscle from the injection site, blood and liver) and the syringe with fluid were stored for four months in a freezer at -20 degrees C. The initial pancuronium concentrations were 81 ng/mL in blood and 532 ng/g in liver. The analyte was stable when stored at -20 degrees C in blood even up to seven months. In liver samples its concentrations were variable. Pancuronium in blood stored at 20 degrees C underwent degradation very rapidly. After three months of storage these blood samples had concentrations not greater about 10% of the initial value. The degradation patterns of pancuronium depended on temperature and the biological matrix.     

Kinetics of ethanol degradation in forensic blood samples

To determine ethanol in human post-mortem blood samples is problematic, largely due to the inappropriate and variable methods of preserving and storing, which can cause decomposition and loss of alcohol concentration. In this study, four crucial parameters of sample conservation were studied: temperature (T), percentage of air chamber in container (%CA), ethanol concentration in blood and post-mortem time. Blood samples from post-mortem cases were stored under different conditions (ethanol levels were known in all cases); factorial design variables: (%CA) 0, 5, 20, 35, 65%; storage temperature: 25, 4 and -10 degrees C; in a total of 15 experiments. No preserving agent was used in samples. Quantification of ethanol in blood was carried out by gas chromatography with head-space FID detector. Initial ethanol concentration ranged from 0.50 to 4.30 g/L. The kinetics of degradation observed was pseudo-first-order. The parameter that characterised the kinetics of ethanol degradation (k(0)) ranged from (4 x 10(-4) and 5.0 x 10(-1) day(-1)), depending on storage conditions. A strong dependence between ethanol degradation and the content of the air chamber was observed and this dependence was found to be stronger than that between degradation and temperature; there was an experimental relation between (k(0)) and (%CA). Activation energy for different conditions, i.e. 0, 5, 20, 35 and 65 (%CA), were calculated and contour plots were made. A mathematical equation relating air chamber, temperature and ethanol concentration at a certain time was determined. This equation allowed estimation of initial concentrations of ethanol with minimal error. A good correlation between experimental data and data calculated with the equation was obtained (r(2) = 0.9998). The best storage conditions were: 0% CA and storage at -10 degrees C, obtaining an ethanol degradation of 0.01% after 15 days. However, 33% of ethanol degradation was obtained with 35% CA at 25 degrees C after 15 days. This equation is useful in forensic cases in which original concentration of ethanol has to be estimated under different sample storage conditions.     

The effect of sodium fluoride preservative and storage temperature on the stability of 6-acetylmorphine in horse blood, sheep vitreous and deer muscle

This study examined the in vitro stability of 6-acetylmorphine (6AM) in horse blood, sheep vitreous humour (VH) and homogenised deer muscle stored under different storage conditions. The stability of 6AM in horse blood is of interest because many toxicological laboratories utilise this matrix for the preparation of blood calibration and check standards and the latter are typically stored during routine use. Data on the storage stability of 6AM in human VH is extremely limited and no data has been reported in muscle. In the absence of human samples, 6AM stability was demonstrated in sheep vitreous and deer muscle. Blood and VH were stored with and without NaF at room temperature (RT), 4 and -18°C for 84 days. Muscle tissue homogenates were prepared in water with and without NaF and also in phosphate buffer (pH 6.0) containing NaF. Homogenates were stored for 31 days at RT, 4 and -18°C. Morphine and 6AM were extracted using SPE and quantified by GC-ion trap-MS/MS. In the absence of NaF, 6AM could not be detected after 7 and 14 days in blood stored at RT and 4°C, respectively. Although at -18°C 6AM was stable for 7 days (12% loss), only 54% was detected by day 84. The addition of NaF to horse blood increased 6AM stability substantially at every temperature. Further, the rate of degradation was found to be significantly slower in blood preserved with 2% NaF compared with 1% NaF (p=.05). 6AM was stable for the study period in preserved blood (1 and 2% NaF) stored at -18°C. For laboratories utilising horse blood in the preparation of standards, preservation with 1% NaF (minimum) and storage at -18°C is recommended. The addition of NaF to VH was essential for 6AM stability. Irrespective of temperature substantial losses (≥ 42%) were observed in unpreserved sheep VH by day 7. In preserved VH the concentration declined by only 22% on day 7 following storage at RT and no loss observed in VH stored at 4 and -18°C at the same time. In muscle, 6AM was stable for 7 days in preserved samples stored at RT and in all samples stored at 4°C and below. The addition of NaF increased the stability of 6AM substantially in muscle. The increased stability of 6AM in VH and muscle preserved with fluoride was attributed to inhibition of bacterial action and the subsequent reduction in the rate of putrefaction of these tissues.     

Stability of diazepam in blood samples at different storage conditions and in the presence of alcohol

Diazepam is one of the mostly used benzodiazepines and it is frequently analyzed in different biological samples, especially blood samples. The diazepam stability in the sample matrices is an important factor regarding reliable data obtaining. The storage is the main factor determining the stability of diazepam in blood samples and it is the object of the study presented. Remaining diazepam amount in spiked whole blood and plasma samples were tested at different storage temperatures, in the absence or presence of sodium fluoride as stabilizer as well as the influence of ethanol on diazepam stability was evaluated. The results of the study indicated that the temperature is the main storage factor affecting diazepam stability. In the fluoride stabilized blood samples the amount of diazepam decreases up to 85% of initial level when stored at -20° C for the period of testing (12 weeks). The presence of low (0.5 g/L) or high (3g/L) ethanol concentrations influences the stability of diazepam at -20 °C. In whole blood samples, the combination of sodium fluoride and ethanol decreases additionally (15-25%) the concentration of the analyte. Freeze-thaw experiments of whole blood samples show about 5-9% decrease in diazepam concentration after the first cycle. The freeze-thaw experiments on plasma samples, containing ethanol and/or fluoride show insignificant decreases of analyte concentration. Further experiments on benzodiazepines stability at different storage conditions or in combination of different factors should be undertaken in forensic toxicology to ensure the data quality, their reliability and reproducibility.     

The effect of sodium fluoride preservative and storage temperature on the stability of cocaine in horse blood, sheep vitreous and deer muscle

The in vitro stability of cocaine in horse blood, sheep vitreous humour (VH) and homogenised deer muscle is described. The stability of cocaine in horse blood was of interest because many toxicology laboratories utilise horse blood for the preparation of calibration and check standards and the latter are typically stored during routine use. The storage stability of cocaine in human VH and muscle has not been previously reported. In the absence of blank human VH and muscle, cocaine stability under varying conditions was demonstrated in animal tissues. Blood and VH were stored with and without addition of NaF at room temperature (RT), 4°C and -18°C for 84 days. Muscle homogenates were prepared in water, water/2% NaF, and phosphate buffer (pH 6.0)/2% NaF, and stored for 31 days at RT, 4°C and -18°C. Cocaine stability in human muscle obtained from cocaine positive forensic cases was assessed following storage at -18°C for 13 months. Cocaine and benzoylecgonine (BZE) were extracted using SPE and quantified by GC-MS/MS. Cocaine was stable for 7 days in refrigerated (4°C) horse blood fortified with 1 and 2% NaF. In the absence of NaF, cocaine was not detectable by day 7 in blood stored at RT and 4°C and had declined by 81% following storage at -18°C. At 4°C the rate of cocaine degradation in blood preserved with 2% NaF was significantly slower than with 1% NaF. The stability of cocaine in horse blood appeared to be less than that reported for human blood, probably attributable to the presence of carboxylesterase in horse plasma. Cocaine stored in VH at -18°C was essentially stable for the study period whereas at 4°C concentrations decreased by >50% in preserved and unpreserved VH stored for longer than 14 days. Fluoride did not significantly affect cocaine stability in VH. The stability of cocaine in muscle tissue homogenates significantly exceeded that in blood and VH at every temperature. In preserved and unpreserved samples stored at 4°C and below, cocaine loss did not exceed 2%. The increased stability of cocaine in muscle was attributed to the low initial pH of post-mortem muscle. In tissue from one human case stored for 13 months at -18°C the muscle cocaine concentration declined by only 15% (range: 5-22%). These findings promote the use of human muscle as a toxicological specimen in which cocaine may be detected for longer compared with blood or VH.     

血液中乙醇保存稳定性研究

目的确定血液中乙醇最佳保存条件,探讨影响血液中乙醇含量稳定性的主要因素。方法对血液保存的温度(-20、4、20℃)、防腐剂(NaF、无防腐剂、Na2O2)、储存容器中空气所占比例(0%、25%、50%)和血醇质量浓度(0.2、0.8、2.0mg/mL)四个因素采用正交试验L9(34)方法分组,样本采用顶空气相色谱法进行测定,测定结果采用方差分析进行讨论。结果在20℃保存且不加入防腐剂的两组样本中血醇浓度变化明显,其余变化不明显。结论血液样本在4℃、储存容器中空气比例为50%和加防腐剂(NaF)的条件下保存,稳定性最佳;四个影响因素中温度为影响血液中乙醇含量稳定性的主要因素。     

Cyanide distribution in five fatal cyanide poisonings and the effect of storage conditions on cyanide concentration in tissue

The cyanide distribution in five fatal cyanide poisonings was analyzed by the pyridine-pyrazolone method using a Conway diffusion cell. In order to study the effect of storage conditions on cyanide concentration in tissue samples, the cyanide concentrations were first measured immediately after collection of the samples at autopsy, then measured again after storage in a refrigerator (4 degrees C) or in a freezer (-20 degrees C) for periods ranging from 1 day to 3 weeks. Concentrations in all but three of the blood samples stored at 4 degrees C or -20 degrees C increased, with concentration ratios based on measurement made before and after storage ranging from 0.71 to 1.46. The concentrations in the liver, kidney, and brain samples either increased or decreased, with ratios of from 0.2 to 8.8. The concentrations in the stomach contents samples decreased rapidly at 4 degrees C, but hardly changed at all at -20 degrees C.     

血中碳氧血红蛋白饱和度测定影响因素的研究

目的 考察血中碳氧血红蛋白饱和度(HbCO％)测定的影响因素,为其结果评定和所需样品保存条件提供实验依据。方法 利用三种分光光度法,测定30d内不同条件下保存的CO阳性血的HbCO％的变化。结果 还原双波长法、双波长法测定结果比较稳定,单波长法抗干扰能力较差;尸检所取血样的保存条件包括温度、保存时间及与空气接触程度对HbCO％的测定均有影响,其中温度影响较为显著。结论 利用还原双波长法与双波长法,并结合光谱扫描观察峰形变化可得到比较可靠的结果。30d内4℃条件下,密闭容器中血样接触少量空气不影响其HbCO％的测定。     

What happens in freezing bodies? Experimental study of histological tissue change caused by freezing injuries.

In order to evaluate histological features of freezing damages to human tissue after death, we froze samples of liver and heart tissue to temperatures of -12 degrees C, -28 degrees C and -80 degrees C, and stored them for 24 and 72 h, respectively, at those temperatures. After thawing and routine preparation for histology, the samples were evaluated both by microscope and with an electronic image analyzer. In all cases, we found extended extracellular spaces and shrunken cells resulting from the freeze-thaw cycle. These features were more pronounced in tissues stored for longer durations. Such findings seem to be typical of tissue that has been frozen prior to examination. Two cases of dead bodies found outdoors at subzero temperatures demonstrate that formerly frozen and unfrozen tissues can be distinguished histologically. The findings are examined in relation to the fundamental laws of cryobiology.     

Optimal storage conditions for highly dilute DNA samples: a role for trehalose as a preserving agent

DNA extraction from trace samples or noninvasively collected samples often results in the recovery of low concentration solutions of DNA that are prone to DNA degradation or other loss. Because of the difficulty in obtaining such samples, and their potentially high value in wildlife and forensic studies, it is critical that optimal methods are employed for their long-term storage. We assessed the amplification yield of samples kept under different storage conditions with the addition of potential preserving agents. We stored dilutions of known concentration human placental DNA, and gorilla fecal DNA, under four conditions (+4 degrees C, -20 degrees C, -80 degrees C, dry at room temperature), and with three additives (Tris EDTA (TE) buffer, Hind III digested Lambda DNA, trehalose). The effectiveness of the treatment methods was tested at regular intervals using qPCR to assess the quantity of amplifiable DNA, and a PCR assay of a larger 757 bp fragment to evaluate the quality of that remaining DNA. The highest quantity of DNA remained in samples stored at -80 degrees C, regardless of storage additives, and those dried at room temperature in the presence of trehalose. Surprisingly, DNA quality was best preserved in the presence of trehalose, either dried or at -80 degrees C; significant quality loss occurred with -20 degrees C and +4 degrees C storage.     

The influence of storage temperature and chemical preservation on the stability of succinylcholine in canine tissue

Succinylcholine (SCh) has been detected six months postmortem in liver, kidney, and injection site muscle of rats given 10 to 200 mg/kg by intramuscular injection. SCh stability was studied in canine tissue to evaluate three storage temperatures and two chemical preservatives at three time periods after injection. Nine mongrel dogs weighing 17.2 to 28 kg were divided equally into three groups and administered either 0.5, 1.0, or 5.0 mg SCh/kg intravenously into the cephalic vein. Liver, kidney, and gastrocnemius muscle were removed 90 min post-injection and divided into twelve portions. Each portion was treated with embalming fluid, physostigmine, the combination (50/50), or nothing. Chemically treated tissues and nontreated tissues were then stored at either 27, 5, or -20 degrees C for a period of up to forty days. Tissue portions were analyzed using ion-pair extraction, chemical demethylation, and gas chromatography with nitrogen phosphorous detection. Stability of SCh was greatest for samples stored at -20 degrees C and preserved with the combination of embalming fluid plus physostigmine. Kidney concentrations of SCh were significantly higher than those in liver or muscle at all doses. SCh was detected 24 h post-injection in all cases. By 40 days, only trace amounts of SCh, if any, could be detected in samples stored at room temperature with no chemical preservatives.     

The effect of temperature on Y-chromosome detection in blood stains

The present investigation is an extension of an earlier work on the effect of various temperatures on Y-chromosome detection. The deteriorating effect of storage at 53 °C was again demonstrated. Liquid blood samples stored at 5 °C were better preserved with regard to Y-chromosomes than those stored at room temperature. This was in conflict with the earlier results on blood stains. New experiments were therefore performed. The results varied. It is suggested that this variation was due to low temperatures having two contrary effects on the results: the well-known preserving effect on biological material and a previously described constricting effect on the heterochromatic areas of the chromosomes making Y-body detection more difficult.     

Secreted blood group substances: distributions in semen and stabilities in dried semen stains

A sensitive microplate hemagglutination-inhibition technique has been used to ascertain the distributions of secreted blood group substances (BGS) in a population of 176 semen specimens and to characterize the stability of these substances in dried semen stains. The BGS concentrations in semen were found to vary throughout a wide range of titer. Despite this latitude of variation, the titers for the component BGS within the blood groups could be described by a log-normal distribution function. Studies of a number of sequential semen specimens obtained from the same donors revealed that the intraindividual variation in BGS titers was much more limited than the interindividual BGS titers. Attempts to correlate variations in titers between A and H in Group A semen or B and H in Group B semen indicated that the levels of these component substances vary independently. Studies of the stability of BGS in Groups A and O semen suggested that these substances were stable when the semen stains were stored at -20 degrees C, 4 degrees C, or at ambient laboratory temperature in a dry state. In contrast, stains stored at 37 degrees C under humid conditions suffered a dramatic loss in BGS titer, with the half-life of the BGS being on the order of 30 days.