What factors contribute to hospital variation in obstetric transfusion rates?

BACKGROUND AND OBJECTIVES: To explore variation in red blood cell transfusion rates between hospitals, and the extent to which this can be explained. A secondary objective was to assess whether hospital transfusion rates are associated with maternal morbidity. MATERIALS AND METHODS: Linked hospital discharge and birth data were used to identify births (n = 279 145) in hospitals with at least 10 deliveries per annum between 2008 and 2010 in New South Wales, Australia. To investigate transfusion rates, a series of random-effects multilevel logistic regression models were fitted, progressively adjusting for maternal, obstetric and hospital factors. Correlations between hospital transfusion and maternal, neonatal morbidity and readmission rates were assessed. RESULTS: Overall, the transfusion rate was 1.4% (hospital range 0.6-2.9) across 89 hospitals. Adjusting for maternal casemix reduced the variation between hospitals by 26%. Adjustment for obstetric interventions further reduced variation by 8% and a further 39% after adjustment for hospital type (range 1.1-2.0%). At a hospital level, high transfusion rates were moderately correlated with maternal morbidity (0.59, P = 0.01), but not with low Apgar scores (0.39, P = 0.08), or readmission rates (0.18, P = 0.29). CONCLUSION: Both casemix and practice differences contributed to the variation in transfusion rates between hospitals. The relationship between outcomes and transfusion rates was variable; however, low transfusion rates were not associated with worse outcomes.

Methods of classification for women undergoing induction of labour: a systematic review and novel classification system.

BACKGROUND: A lack of reproducible methods for classifying women having an induction of labour (IOL) has led to controversies regarding IOL and related maternal and perinatal health outcomes. OBJECTIVES: To evaluate articles that classify IOL and to develop a novel IOL classification system. SEARCH STRATEGY: Electronic searches using CINAHL, EMBASE, WEB of KNOWLEDGE, and reference lists. SELECTION CRITERIA: Two reviewers independently assessed studies that classified women having an IOL. DATA COLLECTION AND ANALYSIS: For the systematic review, data were extracted on study characteristics, quality, and results. Pre-specified criteria were used for evaluation. A multidisciplinary collaboration developed a new classification system using a clinically logical model and stakeholder feedback, demonstrating applicability in a population cohort of 909 702 maternities in New South Wales, Australia, over the period 2002-2011. MAIN RESULTS: All seven studies included in the systematic review categorised women according to the presence or absence of varying medical indications for IOL. Evaluation identified uncertainties or deficiencies across all studies, related to the criteria of total inclusivity, reproducibility, clinical utility, implementability, and data availability. A classification system of ten groups was developed based on parity, previous caesarean, gestational age, number, and presentation of the fetus. Nulliparous and parous women at full term were the largest groups (21.2 and 24.5%, respectively), and accounted for the highest proportion of all IOL (20.7 and 21.5%, respectively). AUTHOR'S CONCLUSIONS: Current methods of classifying women undertaking IOL based on medical indications are inadequate. We propose a classification system that has the attributes of simplicity and clarity, uses information that is readily and reliably collected, and enables the standard characterisation of populations of women having an IOL across and within jurisdictions.

Variation in hospital caesarean section rates and obstetric outcomes among nulliparae at term: a population-based cohort study.

OBJECTIVE: To explore the variation in hospital caesarean section (CS) rates for nulliparous women, to determine whether different case-mix, labour and delivery, and hospital factors can explain this variation and to examine the association between hospital CS rates and outcomes. DESIGN: Population-based cohort study. SETTING: New South Wales, 2009-2010. POPULATION: Nulliparous women with singleton cephalic live births at term. METHODS: Random effect multilevel logistic regression models using linked hospital discharge and birth data. MAIN OUTCOME MEASURES: Prelabour and intrapartum CS rates following spontaneous labour or labour induction; maternal and neonatal severe morbidity rates. RESULTS: Of 67 239 nulliparous women, 4902 (7.3%) had a prelabour CS, 39 049 (58.1%) laboured spontaneously, and 23 288 (34.6%) had labour induced. Overall, there were 18 875 (28.1%) CSs, with labour inductions twice as likely to result in an intrapartum CS compared with women with a spontaneous onset of labour (34.0% versus 15.5%). After adjusting for differences in case-mix, labour and delivery, and hospital factors, the overall variation in CS rates decreased by 78% for prelabour CSs, 52% for intrapartum CSs following spontaneous labour and 9% following labour induction. Adjusting for labour and delivery practices increased the unexplained variation in intrapartum CSs. The adjusted rates of severe maternal and neonatal morbidity were not significantly different across CS rate quintile groups, except for women in spontaneous labour, where the hospitals in the lowest CS quintile had the lowest neonatal morbidity rate. CONCLUSIONS: Differences in clinical practice were substantial contributors to variation in intrapartum CS rates. Our findings suggest that CS rates in some hospitals could be lowered without adversely affect pregnancy outcomes.

Effect of ethanol and methanol on growth of ruminal bacteria Selenomonas ruminantium and Butyrivibrio fibrisolvens.

The effect of ethanol and methanol on growth of several ruminal bacterial strains was examined. Ethanol concentrations as low as 0.2% had a significant, but moderate, inhibitory effect on lag time or growth over time and 3.3% ethanol significantly inhibited maximum optical density obtained by both Selenomonas ruminantium and Butyrivibrio fibrisolvens. Little growth of either strain occurred at 10% ethanol concentrations. Methanol concentrations below 0.5% had little effect on either growth or maximum optical density of Selenomonas ruminantium whereas methanol concentrations below 3.3% had little effect on growth or maximum optical density of Butyrivibrio fibrisolvens. Higher methanol concentrations increasingly inhibited growth of both strains and no growth occurred at a 10% methanol concentration. Concentrations of ethanol or methanol used to add hydrophobic compounds to culture media should be kept below 1%.

Sugar phosphorylation activity in ruminal acetogens.

Acetogenic bacteria Acetitomaculum ruminis, Acetobacterium woodii, and Eubacterium limosum were compared for phosphoenolpyruvate (PEP) and ATP-dependent phosphorylation of glucose and 2-deoxy-glucose. Rate of phosphorylation activity was measured in toluene-treated acetogenic cells using PEP and ATP and radiolabled glucose or 2-deoxy glucose. Eubacterium limosum, most likely has a glucose phosphotransferase system (PTS). In contrast, A. ruminis, and A. woodii had PEP-dependent glucose phosphorylation rates very similar to control rates, suggesting the lack of PTS activity. These results were confirmed by PEP dependent 2-deoxyglucose phosphorylation data. The rates of ATP-dependent glucose phosphorylation were higher than PEP-dependent glucose dependent in all organisms surveyed. Only E. limosum appeared to have PTS. The presence of PTS in E. limosum could explain why it is not capable of utilizing sugars and H(2)/CO(2) simultaneously and why acetogenesis is not as prominant in the rumen because of the availability of carbohydrates as alternative energy substrates.

Dietary fiber content influences soluble carbohydrate levels in ruminal fluids.

The soluble carbohydrate concentration of ruminal fluid, as affected by dietary forage content (DFC) and/or ruminally undegradable intake protein content (UIPC), was determined. Four ruminally cannulated steers, in a 4 × 4 Latin square design, were offered diets containing high (75 % of DM) or low (25 % of DM) DFC and high (6 % of DM) or low (5 % of DM) UIPC, in a 2 × 2 factorial arrangement. Zinc-treated SBM was the primary UIP source. Soluble hexose concentration (145.1 µM) in ruminal fluid (RF) of steers fed low DFC diets exhibited a higher trend (P = 0.08) than that (124.5 µM) of steers fed high DFC diets. UIPC did not modulate (P = 0.54) ruminal soluble hexose concentrations. Regardless of diet, soluble hexose concentration declined immediately after feeding and did not rise until 3 h after feeding (P < 0.0001). Cellobiose (≈90 %) and glucose (≈10 %) were the major soluble hexoses present in RF. Maltose was not detected. Soluble glucose concentration (13.0 µM) was not modified by either UIPC (P = 0.40) nor DFC (P = 0.61). However, a DFC by post-prandial time interaction was detected (P = 0.02). Pentose concentrations were greater (P = 0.02) in RF of steers fed high DFC (100.2 µM) than steers fed low DFC (177.0 µM). UIPC did not influence (P = 0.35) soluble pentose concentration. The identity of soluble pentoses in ruminal fluid could not be determined. However, unsubstituted xylose and arabinose were excluded. These data indicate that: (i) soluble carbohydrate concentrations remain in ruminal fluid during digestion and fermentation; (ii) slight diurnal changes began after feeding; (iii) DFC influences the soluble carbohydrate concentration in RF; and (iv) UIPC of these diets does not affect the soluble carbohydrate concentration of RF.

Patterns of transfer in labour and birth in rural New Zealand.

INTRODUCTION: For many women, and particularly rural women, birthing locally and within their own community is important for personal, social and/or cultural reasons. If concerns about the woman or her baby mean transfer to a secondary or tertiary facility is necessary, this can be disruptive and stressful, especially if road transfer is complicated by terrain, weather or distance, as is often the case in rural New Zealand. The objective of this study was to explore the number of and reason for transfers during labour and birth for well women, close to full term, from primary rural maternity facilities to specialist care in rural New Zealand. METHODS: This retrospective survey of 45 rural maternity units in the North and South Islands of New Zealand was conducted over a 2 year period ending on 30 June 2006. The participants were the 4678 women who began labour in a rural facility during this time period. RESULTS: The survey response rate was 66.6%. The data revealed that 16.6% of women who commenced labour in a rural unit were transferred in labour or within 6 hours of birth; 3% of babies born in rural units were transferred after birth and up to 7 days post-birth. The primary reason for maternal transfer was slow progress in labour (49.67%). Of the 123 babies transferred, this was most often due to respiratory problems (43%). Key features of the rural context (times and distances to be travelled, geological and climatic characteristics, types of transport systems and availability of local assistance) influenced the timeliness of the decision to transfer. CONCLUSIONS: Within New Zealand's regionalised perinatal system, midwives make cautious decisions about transfer, taking into account the local rural local circumstances, and also the topography as it impacts on transport.

Dilution rates influence ammonia-assimilating enzyme activities and cell parameters of Selenomonas ruminantium strain D in continuous (glucose-limited) culture.

AIMS: The objective of this study was to examine the effect of dilution rates (Ds, varying from 0.05 to 0.42 h(-1)) in glucose-limited continuous culture on cell yield, cell composition, fermentation pattern and ammonia assimilation enzymes of Selenomonas ruminantium strain D. METHODS AND RESULTS: All glucose-limited continuous culture experiments were conducted under anaerobic conditions. Except for protein, all cell constituents including carbohydrates, RNA and DNA yielded significant cubic responses to Ds with the highest values at Ds of either 0.10 or 0.20 h(-1). At Ds higher than 0.2 h(-1), fermentation acid pattern shifted primarily from propionate and acetate to lactate production. Succinate also accumulated at the higher Ds (0.30 and 0.42 h(-1)). Glucose was most efficiently utilized by S. ruminantium D at 0.20 h(-1) after which decreases in glucose and ATP yields were observed. Under energy limiting conditions, glutamine synthetase (GS) and glutamate dehydrogenase (GDH) appeared to be the major enzymes involved in nitrogen assimilation suggesting that other potential ammonia incorporating enzymes were of little importance in ammonia assimilation in S. ruminantium D. GS exhibited lower activities than GDH at all Ds, which indicates that the bacterial growth rate is not a primary regulator of their activities. CONCLUSIONS: Studied dilution rates influenced cell composition, fermentation pattern and nitrogen assimilation of S. ruminantium strain D grown in glucose-limited continuous culture. SIGNIFICANCE AND IMPACT OF THE STUDY: Selenomonas ruminantium D is an ecologically and evolutionary important bacterium in ruminants and is present under most rumen dietary conditions. Characterizing the growth physiology and ammonia assimilation enzymes of S. ruminantium D during glucose limitation at Ds, which simulate the liquid turnover rates in rumen, will provide a better understanding of how this micro-organism responds to differing growth conditions.

Collaboration across the pond: the multi-school progress testing project.

This collaborative project between the National Board of Medical Examiners and four schools in the UK is investigating the feasibility and utility of a cross-school progress testing program drawing on test material recently retired from the United States Medical Licensing Examination (USMLE) Step 2 Clinical Knowledge (CK) examination. This article describes the design of the progress test; the process used to build, translate (localize), review, and finalize test forms; the approach taken to (web-based) test administration; and the procedure used to calculate and report scores. Results to date have demonstrated that it is feasible to use test items written for the US licensing examination as a base for developing progress test forms for use in the UK. Some content areas can be localized more readily than others, and care is clearly needed in review and revision of test materials to ensure that it is clinically appropriate and suitably phrased for use in the UK. Involvement of content experts in review and vetting of the test material is essential, and it is clearly desirable to supplement expert review with the use of quality control procedures based on the item statistics as a final check on the appropriateness of individual test items.

The influence of copper concentration and source on ileal microbiota.

Copper is normally supplemented in poultry diets as a growth promotant and antimicrobial. However, there are conflicting reports about the growth benefits and little information about how Cu affects the microbiota in the intestinal tract of poultry. Therefore, in vitro and in vivo experiments were conducted with broilers to determine the effects of Cu source and supplementation on ileal microbiota. The influence of Cu on growth of lactobacilli and Escherichia coli in media inoculated with ileal contents was determined in the first study. When Cu sulfate pentahydrate was supplemented to the cultures, quadratic increases in lactobacilli to graded concentrations of Cu up to 125 mg/kg and quadratic decreases in E. coli up to 250 mg/kg of Cu were observed after 24 h of incubation at 37 degrees C. However, when tribasic Cu chloride (TBCC) was supplemented, neither linear nor quadratic responses to graded concentrations of dietary Cu were observed on number of lactobacilli or number of E. coli. The effects of Cu and Cu source on ileal microbiota and growth performance in broiler chickens were determined in the second study. Bird performance was not affected by Cu source or concentration. The bacterial culture enumeration results revealed that supplementation with 187.5 mg/kg of Cu from Cu sulfate pentahydrate and TBCC had no effect on number of ileal lactobacilli of birds. The denaturing gradient gel electrophoresis analyses of ileal microbial communities revealed that neither Cu supplementation nor source had effects on the number of bacterial species predominant in the ileal digesta or associated with the ileal mucosa. Supplementation with TBCC supplementation significantly increased the similarity coefficients of microbiota in the ileal mucosa compared with cross-products of all individuals. This suggests that TBCC may alter the intestinal microbiota, yet this shift had no effect on bird performance.

Influence of stressors on normal intestinal microbiota, intestinal morphology, and susceptibility to Salmonella enteritidis colonization in broilers.

In modern poultry production systems, environmental stressors may influence bird performance and susceptibility to pathogens such as Salmonella Enteritidis. Experiments were conducted to determine the influence of 24-h feed withdrawal and 24-h exposure to high temperature (30 degrees C) on intestinal characteristics of broilers. Attachment of Salmonella Enteritidis to ileal tissue was determined using an in vitro ileal loop assay. Changes in commensal intestinal microbial populations were determined using denaturing gradient gel electrophoresis, and alterations in ileal morphology were determined histologically. Ex vivo attachment of Salmonella Enteritidis to ileal tissues increased by 1.5 logs (9.05 log10 vs. 7.59 log (10) Salmonella Enteritidis/g of ileal tissue; P = 0.0006) in broilers fasted for 24 h. Similarly, ileal tissues from birds subjected to 30 degrees C for 24 h had increased ex vivo attachment of Salmonella Enteritidis (8.77 log(10) vs. 8.50 log(10) Salmonella Enteritidis/g of ileum; P = 0.01) compared with birds held at 23 degrees C. Exposure to 30 degrees C for 24 h also altered microbial community structure in the ileum and cecum. Subjecting birds to 30 degrees C for 24 h reduced crypt depth (6.0 vs. 7.8 microm, respectively; P = 0.002), but had no effect on villus height or villus:crypt ratio. This research shows that acute stressors in poultry production systems can cause changes in the normal intestinal microbiota and epithelial structure, which may lead to increased attachment of Salmonella Enteritidis.

Performance and nutrient retention responses of broilers to dietary oxyhalogenic and ionic salts.

Two experiments were conducted to determine the effects of 2 ionic and antimicrobial mixtures on broiler performance and nutrient retention. In experiment 1, male broilers were fed 6 diets in a 2 x 3 factorial experiment (5 cages/diet, 9 chicks/cage) from 0 to 21d of age. Diets with 2 nutrient densities [normal industry diet (ND) and a low nutrient density diet (82% of ND)] and 3 ionic and antimicrobial mixtures [none (control) or 1 of 2 formulations containing different mixtures of ionic salts and oxyhalogenic compounds (sodium salts of chlorite, chlorate, chloride, borate, sulfate, bromide, salicylate, and hydrogen peroxide) at 4.4 mL/kg of feed (mix A and B)]. Birds fed mix B (568.6 g) were heavier (P < 0.05) at 21d of age than birds fed the control diet (501.7 g) and BW of birds fed mix A (536.1 g) did not differ from mix B or controls. Phosphorus and nitrogen retention from 18 to 20 d in birds fed mix B (78.05% and 82.23%, respectively) was greater (P < 0.05) than birds fed mix A (60.21 and 71.22%, respectively) and birds fed mix A had greater (P < 0.05) retention than birds fed the control diet (45.94 and 69.06%, respectively). In experiment 2, chicks were fed either 4.4 mL of mix B/kg feed, a diet with salinomycin and bacitracin, or a control diet. Birds fed the control or mix B diet had greater (P < 0.05) BW at 18 d than birds on the antibiotic treatment, whereas diet or nutrient retention differences were not present at 42 d of age. In conclusion, the ionic and antimicrobial mixtures improved performance and nutrient retention in young broilers but these did not last until market age.

Effects of delayed placement on intestinal characteristics in turkey poults.

To determine the effects of delayed access to feed on intestinal villus development, bacterial translocation, and salmonella attachment potential in turkey poults, poults were given ad libitum access to water and feed immediately after hatch (FED) or were delayed access to water and feed for 48 h (DLY). In experiment 1, FED poults were heavier until 5 d postfeeding at which time villi were 50 microm longer and 6.8 microm wider, crypts were 5.9 microm deeper, and there were more goblet cells per villus than in the DLY poults. The DLY poults also had reduced numbers of proliferating enterocytes in the villus and higher apoptotic labeling at 1, 2, and 5 d postfeeding. In experiment 2, DLY poults had higher numbers of aerobic bacteria in the ileal digesta and ileal tissue when compared with the FED poults at 1 d postfeeding. To study salmonella attachment potential to the ileum, a segment of the ileum was inoculated with a salmonella culture, incubated for 1 h at 37 degrees C, and flushed; the tissue was homogenized, and numbers of remaining salmonella were enumerated. No differences in salmonella attachment potential were noted between FED and DLY poults. In conclusion, early access to ad libitum feed in poults stimulated growth and development of small intestinal villi and their absorptive surfaces, whereas delayed access to feed resulted in delayed enterocyte proliferation and greater enterocyte apoptosis during the first week posthatch as well as greater numbers of aerobic bacteria associated with the small intestine shortly after hatch.

Monoclonal antibody 4F2 reactive with basal layer keratinocytes: studies in the normal and a hyperproliferative state.

To establish a method for separating different keratinocyte subpopulations in the epidermis, we studied the specificity of monoclonal antibody 4F2 for keratinocytes. Preliminary screening experiments had previously demonstrated 4F2 reactivity with the epidermis. 4F2 reacted with a subpopulation (19.29 +/- 5.23%) of human epidermal cells in suspension. The membrane antigen identified by 4F2 continues to be expressed by cultured keratinocytes. In frozen tissue section using an indirect immunofluorescence technique, the 4F2-positive cells in the basal layer are sharply demarcated from the negative suprabasilar layers. Even in the hyperproliferative state of psoriasis, the 4F2 reactivity is confined to the basal layer. Cell suspensions of psoriatic epidermis demonstrated a greater percentage of reactivity with 4F2 (49.51% +/- 6.50%), probably reflecting the expanded population of basal layer cells. Monoclonal 4F2, therefore, reacts with a membrane antigen present on basal keratinocytes, and provides a probe for use in the isolation of the basal keratinocyte subpopulation. Thus, this antibody should be useful in studies of normal and aberrant differentiation of the epidermis.

Thymopoietin-like substance in human skin.

A heterologous antithymopoietin (anti-TP) antibody was used to determine whether a TP-like molecule is present in the epidermis, since such factors have been postulated to play a part in known T cell-epidermal cell interaction. Examination of cytocentrifuge smears of freshly separated human epidermal cells stained by indirect immunofluorescence revealed that 8-14% of these cells possessed cytoplasmic reactivity with the anti-TP antibody. Similarly, 2-5% of human epidermal cells, maintained in tissue culture for 2-8 weeks, showed cytoplasmic staining with the anti-TP antibody. Double-labeling immunofluorescence studies, with the anti-TP antibody and a monoclonal antibody specifically reactive with Langerhans cells (OKT6), demonstrated that cells possessing this TP-like substance were not Langerhans cells. In situ studies of 4-microns frozen sections of normal human skin indicated that the cell population which possesses the TP-like substance is the basal layer of keratincoytes in the epidermis.

Kaposi's sarcoma following chemotherapy for testicular cancer in a homosexual man: demonstration of cytomegalovirus RNA in sarcoma cells.

A 35-year-old white Jewish homosexual man who had undergone surgery and chemotherapy for an embryonal carcinoma of the testis subsequently developed Kaposi's sarcoma. The neoplasm involved the skin as well as visceral tissues. Tissue derived from a biopsy specimen of one of the skin lesions was used in the in situ hybridization technique for the detection of genetic material. Cytomegalovirus messenger RNA was identified in the neoplastic Kaposi cells in the skin. The significance of this finding is discussed.

Comparison of Maxon suture with Vicryl, chromic catgut, and PDS sutures in fascial closure in rats.

Two hundred ten rats were randomized into one of five study groups to compare standard absorbable sutures with a new synthetic absorbable suture. We evaluated Maxon (polyglyconate), Vicryl (polyglactin), chromic catgut (catgut), and PDS (polydioxanone) with respect to tissue inflammatory reaction, knot security, suture tensile strength, and suture absorption. The results indicate that Maxon and PDS elicited a lower degree of chronic inflammation when compared with Vicryl and chromic catgut. The tensile strengths of Maxon and Vicryl significantly exceeded those of PDS and chromic catgut during the critical period of wound healing. Maxon and PDS retained a larger percentage of tensile strength during the long postoperative period, whereas Vicryl and chromic catgut were mostly absorbed. Maxon is an excellent addition to the armamentarium of the gynecologic surgeon.

Pulmonary venoocclusive disease in pregnancy.

A 23-year-old woman in her 27th week of gestation presented with clinical findings of progressive pulmonary hypertension. After cardiac catheterization she went into labor and was delivered by cesarean section. She died shortly thereafter from right heart failure. Pulmonary venoocclusive disease was found at autopsy. Hemodynamic changes during pregnancy, labor, delivery, and the postpartum period may have contributed to her deterioration and death. This is the first description of pulmonary venoocclusive disease in pregnancy.

Correlation of cellular atypia and human papillomavirus deoxyribonucleic acid sequences in exfoliated cells of the uterine cervix.

Restriction enzyme digestion and Southern blot hybridization were used to analyze deoxyribonucleic acid (DNA) extracted from exfoliated cervical cells for the presence of human papillomavirus sequences and these results were correlated with cytologic findings on Papanicolaou smears. Specimens (N = 204) were obtained from a nonselected population of women undergoing routine cytologic screening and human papillomavirus DNA sequences were detected in 33 (16%) women. Thirteen smears contained atypical squamous cells, ranging from very mild dysplasia to moderate dysplasia; all showed associated morphologic evidence of human papillomavirus infection characterized by koilocytosis, nuclear enlargement, wrinkling, and hyperchromasia, and human papillomavirus DNA was demonstrable in 12 (92%) smears. Of the remaining 191 samples with normal cytology, 21 (11%) also contained human papillomavirus DNA sequences. Reevaluation of the smears from these women resulted in a revision of the cytologic diagnosis to very mild dysplasia in four cases. These data suggest that human papillomavirus infection occurs more frequently than predicted by cytologic screening.

A modified, prefixed, Golgi-rapid technique for the cephalopod retina.

A quick and simple method for Golgi staining of neurons and glia in the cephalopod retina is described. The main difference between this method and other Golgi-rapid protocols is that tissue is placed in sachets constructed from dialysis membrane prior to chromation and impregnation. This greatly reduces the amount of surface deposition of silver chromate and can reduce the number of elements impregnated. Together these effects produce improved Golgi staining of the cephalopod retina. Given some modifications to the osmolarity of the solutions employed, the method should be applicable to other, non-cephalopod tissues where superficial structures are obscured by surface precipitation, or neurons are arranged in thin sheets of tissue. The method employs prefixation of tissue in a fixative suitable for light or electron microscopy.