An effective palynological preparation procedure using hydrogen peroxide

Most pre-Quaternary palynology samples are currently prepared by demineralization of the sediment/sedimentary rock matrix using hydrochloric and hydrofluoric acids (HCl and HF respectively). If a consistently effective alternative to this procedure can be developed, palynological processing will be made significantly less hazardous to both laboratory personnel, and to the wider environment. Furthermore, most non-acid processing methods are normally quicker and cheaper than matrix dissolution using acid. Some authors have previously used hydrogen peroxide (H2O2) to extract palynomorphs by the physico–chemical disaggregation of the clay fraction. However, H2O2 is a powerful oxidizing agent and hence can potentially destroy sedimentary organic material, including palynomorphs. A new method using hot H2O2, where exposure of the sample material to the H2O2 is minimized, has been developed. Crushed sample material in a suitable vessel is placed on a hot plate for one minute, treated with 15–30% H2O2 for 10 minutes, then the residue is diluted with cold distilled water. Disaggregated sample material tends to float, and is decanted into a large vessel containing distilled water to further dilute the H2O2. If any undisaggregated sample remains, the procedure is repeated several times if necessary. Relatively indurated sedimentary lithotypes normally require several treatments. The reason for this stepwise treatment is that the organic material is not exposed to H2O2 for sustained periods, thereby reducing the possibility of palynomorph damage/degradation due to oxidation. When the sample matrix has been fully disaggregated, the residue can be further processed as appropriate. In this study, eight samples of Carboniferous, Jurassic, Paleogene, and Quaternary age were prepared quantitatively using the new H2O2 method. These were all prepared using 30% H2O2. For comparison, they were also prepared quantitatively using HCl/HF and/or sodium hexametaphosphate [(NaPO3)6]. Quantitative preparations allow the concentration of palynomorphs extracted to be determined, and therefore the effectiveness of the techniques used can be compared objectively. The palynomorph residues derived from these three techniques varied markedly. The H2O2 method does not consistently disaggregate all the sample material, particularly the older and more indurated lithotypes. Some evidence of oxidation effects was observed. Two samples of Mississippian mudstone from the U.S.A. were prepared using H2O2 and (NaPO3)6. Both methods produced abundant miospores, however the H2O2 procedure yielded far higher palynomorph concentrations than the (NaPO3)6 technique. Minor degradation of palynomorphs in the H2O2 preparation was noted. The H2O2 and HCl/HF methods were compared directly on a palynomorph-rich sample of Upper Carboniferous mudstone from offshore Scotland. Both preparations produced abundant miospores. The HCl/HF method had significantly higher recovery levels than the H2O2 procedure. It appears that the H2O2 method simultaneously macerates the matrix, and oxidizes any amorphous organic material (AOM) present. In this sample, the HCl/HF residue was relatively rich in AOM. By contrast, the H2O2 preparation is virtually clear of this phytoclast type, which partially obscures palynomorphs. Two samples of the Middle Jurassic Grantham Formation of eastern England were processed using H2O2 and HCl/HF. The two methods produced abundant palynofloras of similar palynomorph concentrations. Two dinoflagellate cyst acmes within the Danian (Paleogene) part of the López de Bertodano Formation of Seymour Island, Antarctica were also tested using H2O2, (NaPO3)6, and HCl/HF. TheH2O2 preparation completely destroyed the dominant taxon, Palaeoperidinium pyrophorum, in one sample. By contrast, the (NaPO3)6 and HCl/HF preparations produced abundant, fully representative palynofloras. In the other sample, the acme of Spinidinium spp. is completely unaffected by the H2O2 preparation procedure. The final sample of this study is an unconsolidated clay of Late Pleistocene age from offshore Scotland. Both the H2O2 and HCl/HF preparations proved similar in both taxonomic content and overall palynomorph yield. The new method of preparation using hot H2O2 has proved to be extremely effective. In particular, it appears to be superior to the (NaPO3)6 procedure for indurated lithotypes. However care should be taken because H2O2 can destroy certain dinoflagellate cysts and kerogen macerals which are especially susceptible to oxidation. Further development work, and more comparative testing of the H2O2, (NaPO3)6, and HCl/HF procedures, should be undertaken

Adding 6 months of androgen deprivation therapy to postoperative radiotherapy for prostate cancer: a comparison of short-course versus no androgen deprivation therapy in the RADICALS-HD randomised controlled trial

Background Previous evidence indicates that adjuvant, short-course androgen deprivation therapy (ADT) improves metastasis-free survival when given with primary radiotherapy for intermediate-risk and high-risk localised prostate cancer. However, the value of ADT with postoperative radiotherapy after radical prostatectomy is unclear. Methods RADICALS-HD was an international randomised controlled trial to test the efficacy of ADT used in combination with postoperative radiotherapy for prostate cancer. Key eligibility criteria were indication for radiotherapy after radical prostatectomy for prostate cancer, prostate-specific antigen less than 5 ng/mL, absence of metastatic disease, and written consent. Participants were randomly assigned (1:1) to radiotherapy alone (no ADT) or radiotherapy with 6 months of ADT (short-course ADT), using monthly subcutaneous gonadotropin-releasing hormone analogue injections, daily oral bicalutamide monotherapy 150 mg, or monthly subcutaneous degarelix. Randomisation was done centrally through minimisation with a random element, stratified by Gleason score, positive margins, radiotherapy timing, planned radiotherapy schedule, and planned type of ADT, in a computerised system. The allocated treatment was not masked. The primary outcome measure was metastasis-free survival, defined as distant metastasis arising from prostate cancer or death from any cause. Standard survival analysis methods were used, accounting for randomisation stratification factors. The trial had 80% power with two-sided α of 5% to detect an absolute increase in 10-year metastasis-free survival from 80% to 86% (hazard ratio [HR] 0·67). Analyses followed the intention-to-treat principle. The trial is registered with the ISRCTN registry, ISRCTN40814031, and ClinicalTrials.gov, NCT00541047. Findings Between Nov 22, 2007, and June 29, 2015, 1480 patients (median age 66 years [IQR 61–69]) were randomly assigned to receive no ADT (n=737) or short-course ADT (n=743) in addition to postoperative radiotherapy at 121 centres in Canada, Denmark, Ireland, and the UK. With a median follow-up of 9·0 years (IQR 7·1–10·1), metastasis-free survival events were reported for 268 participants (142 in the no ADT group and 126 in the short-course ADT group; HR 0·886 [95% CI 0·688–1·140], p=0·35). 10-year metastasis-free survival was 79·2% (95% CI 75·4–82·5) in the no ADT group and 80·4% (76·6–83·6) in the short-course ADT group. Toxicity of grade 3 or higher was reported for 121 (17%) of 737 participants in the no ADT group and 100 (14%) of 743 in the short-course ADT group (p=0·15), with no treatment-related deaths. Interpretation Metastatic disease is uncommon following postoperative bed radiotherapy after radical prostatectomy. Adding 6 months of ADT to this radiotherapy did not improve metastasis-free survival compared with no ADT. These findings do not support the use of short-course ADT with postoperative radiotherapy in this patient population

Duration of androgen deprivation therapy with postoperative radiotherapy for prostate cancer: a comparison of long-course versus short-course androgen deprivation therapy in the RADICALS-HD randomised trial

Background Previous evidence supports androgen deprivation therapy (ADT) with primary radiotherapy as initial treatment for intermediate-risk and high-risk localised prostate cancer. However, the use and optimal duration of ADT with postoperative radiotherapy after radical prostatectomy remains uncertain. Methods RADICALS-HD was a randomised controlled trial of ADT duration within the RADICALS protocol. Here, we report on the comparison of short-course versus long-course ADT. Key eligibility criteria were indication for radiotherapy after previous radical prostatectomy for prostate cancer, prostate-specific antigen less than 5 ng/mL, absence of metastatic disease, and written consent. Participants were randomly assigned (1:1) to add 6 months of ADT (short-course ADT) or 24 months of ADT (long-course ADT) to radiotherapy, using subcutaneous gonadotrophin-releasing hormone analogue (monthly in the short-course ADT group and 3-monthly in the long-course ADT group), daily oral bicalutamide monotherapy 150 mg, or monthly subcutaneous degarelix. Randomisation was done centrally through minimisation with a random element, stratified by Gleason score, positive margins, radiotherapy timing, planned radiotherapy schedule, and planned type of ADT, in a computerised system. The allocated treatment was not masked. The primary outcome measure was metastasis-free survival, defined as metastasis arising from prostate cancer or death from any cause. The comparison had more than 80% power with two-sided α of 5% to detect an absolute increase in 10-year metastasis-free survival from 75% to 81% (hazard ratio [HR] 0·72). Standard time-to-event analyses were used. Analyses followed intention-to-treat principle. The trial is registered with the ISRCTN registry, ISRCTN40814031, and ClinicalTrials.gov , NCT00541047 . Findings Between Jan 30, 2008, and July 7, 2015, 1523 patients (median age 65 years, IQR 60–69) were randomly assigned to receive short-course ADT (n=761) or long-course ADT (n=762) in addition to postoperative radiotherapy at 138 centres in Canada, Denmark, Ireland, and the UK. With a median follow-up of 8·9 years (7·0–10·0), 313 metastasis-free survival events were reported overall (174 in the short-course ADT group and 139 in the long-course ADT group; HR 0·773 [95% CI 0·612–0·975]; p=0·029). 10-year metastasis-free survival was 71·9% (95% CI 67·6–75·7) in the short-course ADT group and 78·1% (74·2–81·5) in the long-course ADT group. Toxicity of grade 3 or higher was reported for 105 (14%) of 753 participants in the short-course ADT group and 142 (19%) of 757 participants in the long-course ADT group (p=0·025), with no treatment-related deaths. Interpretation Compared with adding 6 months of ADT, adding 24 months of ADT improved metastasis-free survival in people receiving postoperative radiotherapy. For individuals who can accept the additional duration of adverse effects, long-course ADT should be offered with postoperative radiotherapy. Funding Cancer Research UK, UK Research and Innovation (formerly Medical Research Council), and Canadian Cancer Society

A direct comparison of three palynological preparation techniques

Two samples of palynomorph-rich Upper Jurassic (Lower Oxfordian) mudstone from western Scotland were quantitatively prepared using the traditional hydrochloric and hydrofluoric acid based palynological preparation technique and two non-acid procedures. The latter are protocols using sodium hexametaphosphate [(NaPO3)6] and hydrogen peroxide (H2O2). These non-acid techniques have previously been validated only in terms of the absolute numbers of palynomorphs extracted. By contrast, this study aimed to assess the numbers of palynomorphs extracted in terms of absolute numbers of the individual taxa present to test for any taxonomic biases. The (NaPO3)6 method proved around 50% as efficient as acid digestion in terms of absolute numbers of palynomorphs extracted. It produced clean residues, which are eminently suitable for most palynological studies. The majority of the taxa present were recovered in representative relative proportions, and no taxonomic biases were noted. The absolute numbers of most taxa decrease in a stepwise fashion from acid digestion via the (NaPO3)6 procedure to the H2O2 method. However, the concentrations of bisaccate pollen were apparently relatively unaffected by the three methods used. Similarly, the Meiourogonyaulax caytonensis group appears to be unusually resistant to oxidation damage by H2O2. It is considered that the (NaPO3)6 preparation method is an eminently viable alternative to acid digestion, especially in remote operational settings such as rigsites. The H2O2 technique proved to be significantly less effective, at approximately 10% of the extraction level of acid digestion which appears to be largely due to oxidation. Hydrogen peroxide is an aggressive oxidant. Therefore the (NaPO3)6 technique is deemed to be both safer and more effective than the H2O2 method

The use of pre-treatments in palynological processing

A sample of palynomorph-rich Upper Carboniferous mudstone from Scotland was separately pre-treated overnight with acetone, two detergent solutions, formic acid, household bleach (two methods), methylated spirits and white spirit prior to palynological preparation using sodium hexametaphosphate [(NaPO3)6]. The aim of this study was to identify effective methods of pre-treatment that would increase palynomorph yields using the (NaPO3)6 method. Pre-treatment generally increased the mass of sample that was broken down by the (NaPO3)6 technique. Detergent one (carpet cleaner), formic acid, household bleach and white spirit allowed the disaggregation of more rock than without any pre-treatment. However, formic acid produced a lower concentration of Carboniferous miospores than with no pre-treatment. Pre-treatment with acetone, detergent two (industrial detergent) and methylated spirits actually decreased the weight of rock that was disaggregated with (NaPO3)6. Despite this, all these three pre-treatments improved the palynomorph yield as compared to with no pre-treatment. Moreover, all the pre-treatments except formic acid improved palynomorph productivity. The effectiveness of pre-treatments was demonstrated by the increased absolute numbers of indigenous palynomorphs extracted. However, the concentrations of miospores per gram of rock are more significant. Acetone, both detergent solutions, methylated spirit and white spirit significantly improved the amounts of palynomorph extracted. Household bleach was found to lighten and selectively destroy relatively delicate palynomorphs; this reagent should be used with caution, and only with robust material. In the subsample soaked overnight in 5% bleach solution, all the exotic Lycopodium spores added were destroyed. By contrast in the subsample treated with 2.5% bleach solution for 6h, a small proportion of the exotic Lycopodium spores survived. This study indicates that the (NaPO3)6 method using either detergent or white spirit as a pre-treatment is highly effective at extracting palynomorphs from clay-rich lithotypes. However the concentration of palynomorphs obtained is generally lower than those from mineral acid digestion

Determining the absolute abundance of dinoflagellate cysts in recent marine sediments : the Lycopdium marker-grain method put to the test

Absolute abundances (concentrations) of dinoflagellate cysts are often determined through the addition of Lycopodium clavatum marker-grains as a spike to a sample before palynological processing. An inter-laboratory calibration exercise was set up in order to test the comparability of results obtained in different laboratories, each using its own preparation method. Each of the 23 laboratories received the same amount of homogenized splits of four Quaternary sediment samples. The samples originate from different localities and consisted of a variety of lithologies. Dinoflagellate cysts were extracted and counted, and relative and absolute abundances were calculated. The relative abundances proved to be fairly reproducible, notwithstanding a need for taxonomic calibration. By contrast, excessive loss of Lycopodium spores during sample preparation resulted in non-reproducibility of absolute abundances. Use of oxidation, KOH, warm acids, acetolysis, mesh sizes larger than 15 µm and long ultrasonication (> 1 min) must be avoided to determine reproducible absolute abundances. The results of this work therefore indicate that the dinoflagellate cyst worker should make a choice between using the proposed standard method which circumvents critical steps, adding Lycopodium tablets at the end of the preparation and using an alternative method