Associations between mild-to-moderate anaemia in pregnancy and helminth, malaria and HIV infection in Entebbe, Uganda

Summary
1
Introduction
Anaemia in pregnancy contributes to maternal deaths and may also contribute to adverse birth outcomes including intrauterine growth retardation and prematurity, and hence to perinatal morbidity and mortality (WHO, 1991).
Hookworm is an important cause of anaemia in developing countries, but lack of consensus regarding the risks and benefits of treating helminths in pregnancy has, until recently, led to a tendency to exclude pregnant and even breastfeeding women from deworming programmes. In 1994, a call was made for research leading to improved estimates of hookworm infection in women of child-bearing age and for evaluation of interventions that might be beneficial (WHO, 1994) but, to our knowledge, only one placebo-controlled trial of the treatment of hookworm in pregnancy has been reported (Torlesse and Hodges, 2000, 2001).
More recently, an Informal Consultation held by the WHO gave consideration to the possible effects of schistosomiasis in pregnancy and it was suggested that anaemia might be among them (Allen et al., 2002; WHO, 2002). Although praziquantel had been widely avoided in pregnant and lactating women, it was noted that there was no evidence from studies in animals, from case reports or from mass treatment campaigns in humans of any major adverse effects. Treatment of schistosomiasis during pregnancy was therefore advocated by the Consultation Committee.
We have undertaken a trial designed to examine the effects of maternal helminths and of deworming during pregnancy on the response to immunisation and susceptibility to infection and disease in infancy [ISRCTN32849447] (Elliott et al., 2007). Anaemia is among the secondary outcomes of the trial. The trial is ongoing. In view of current interest in the role of helminths in anaemia during pregnancy, we have examined associations between anaemia and helminths and other major infections (malaria and HIV) among pregnant women at enrolment into the trial.
2
Materials and methods
2.1
Study population and procedures
The study area comprises Entebbe Municipality and the adjacent subcounty of Katabi. This area supports semi-urban, rural and fishing communities residing on the Entebbe peninsula in Lake Victoria, Uganda. Women were recruited at the antenatal clinic at Entebbe Hospital between April 2003 and November 2005.
Women were assessed for screening at their first antenatal visit, thus initial screening could take place in any trimester of pregnancy. They were eligible for screening if they were well, resident in the study area, planning to deliver their baby at the hospital, willing to participate and willing to know their HIV status. On the screening day, after giving written informed consent, eligible women were interviewed regarding sociodemographic characteristics and risk factors for helminth infection, malaria and HIV and were examined by a midwife. A blood sample was obtained for investigations including haemoglobin (Hb) estimation, examination for microfilariae (mf) and malaria parasites, syphilis and HIV serology.
Screened women were asked to return for enrolment within 1 month with a stool sample. They were excluded from enrolment if they had a Hb level <8 g/dl, clinically apparent severe liver disease, diarrhoea with blood in the stool, an abnormal pregnancy, a history of adverse reaction to anthelminthic drugs or had already participated in the study during an earlier pregnancy. Women were enrolled in the trial when they returned with a stool sample if they were in the second or third trimester and full eligibility was confirmed.
All women received routine antenatal care including haematinics and intermittent presumptive treatment for malaria using sulfadoxine/pyrimethamine. Women were treated for syphilis and provided with nevirapine for prevention of mother-to-child HIV transmission, if indicated. Women who were excluded from the study on grounds of severe anaemia were treated with albendazole and haematinics and referred for transfusion if required.
2.2
Haemoglobin estimation and definition of anaemia
Hb was estimated at the antenatal clinic using a colorimetric haemoglobinometer (DHT haemoglobin meter; Developing Health Technology, Barton Mills, UK) with same-day results. The same sample was then sent to the Medical Research Council/Uganda Virus Research Institute (MRC/UVRI) laboratories for analysis by a Coulter analyser (Beckman Coulter AC-T 5 diff CP; Beckman Coulter, Nyon, Switzerland). Quality control for the Coulter analyser was provided through the United Kingdom National External Quality Assessment Schemes, with consistently good results. Initial evaluation suggested that the haemoglobinometer results reliably matched the Coulter analyser results (intraclass correlation coefficient (ICC) = 0.81, 95% CI 0.74–0.88) and the immediately available haemoglobinometer results were used to determine enrolment. Evaluation of results to March 2005 showed less reliability (ICC = 0.54, 95% CI 0.51–0.58) and it was noted that 37 women had been enrolled with haemoglobinometer results >8 g/dl but Coulter analyser values below this cut-off; thereafter, Coulter analyser results were used for enrolment. Coulter analyser results have been used in this analysis.
In accordance with WHO criteria, anaemia was defined as Hb < 11.2 g/dl, i.e. 0.2 g/dl above the standard cut-off of 11 g/dl to allow for the altitude in Entebbe (1132 m a.s.l.) (http://www.sph.emory.edu/∼cdckms/hbadj2.html) (WHO, 1999a).
2.3
Parasitology
Examination of stool samples was performed as described previously (Bukusuba et al., 2004) using the Kato–Katz method (Katz et al., 1972) and charcoal culture for Strongyloides (Friend, 1996). Two Kato–Katz slides were prepared from each sample, each examined within 30 min for hookworm or the following day for other parasites. Blood was examined for Mansonella by a modified Knott's method (Melrose et al., 2000). Intensity of infection was assessed by egg counts in stool and mf counts in blood. Intensities were categorised as follows: hookworm: light <1000 eggs per gram of stool (epg), moderate 1000–3999 epg, high ≥4000 epg (WHO, 1994); Schistosoma mansoni: light <100 epg, moderate 100–399 epg, high ≥400 epg (WHO, 1999b); Trichuris trichiura: light <1000 epg, moderate 1000–9999 epg, high ≥10 000 epg (WHO, 1999b). No standard categories are available for Mansonella intensity, therefore arbitrary categories were defined to obtain approximately equal numbers of participants in each category: light <30 mf/ml, moderate 30–99 mf/ml, high ≥100 mf/ml.
2.4
HIV serology
HIV serology was performed using a rapid test algorithm with same-day results. Testing kits, provided by the Ministry of Health, varied with availability. Most commonly, Determine (Abbott Laboratories, Abbott Japan Co. Ltd., Tokyo, Japan) was used for screening, with positive results confirmed by Unigold (Trinity Biotech plc, Bray, Ireland). Samples with differing results were referred for analysis by non-rapid ELISA tests or were examined using a ‘tie-breaker’ rapid test, usually Statpack (Chembio Diagnostic Systems, Medford, NY, USA). A proportion of samples, including all those with differing results by rapid test, were re-examined at the MRC/UVRI laboratories for quality control, with high agreement of the results.
2.5
Demographic information and potential risk factors for anaemia
Socioeconomic data were summarised by developing two indices based on the variables that appeared to describe socioeconomic status most usefully. These were ‘woman's socioeconomic index’, comprised of education, personal income and occupation, and ‘household socioeconomic index’, comprised of building materials, number of rooms and items collectively owned. The relationship between potential confounding factors and anaemia and helminths, malaria and HIV were considered, and a diagram describing hypothesised relationships was developed (Figure 1). Age, socioeconomic status and tribe were considered to be possible confounders, with gravidity of potential importance, particularly for malaria, to which primigravidae are known to be particularly susceptible.
2.6
Data management and statistical analysis
Data were entered using Microsoft Access (Microsoft Corp., Redmond, WA, USA) and analysed using STATA version 8 (Stata Corp., College Station, TX, USA). The initial analysis was based on a binary variable for outcome (anaemic/not anaemic) and categorical variables for exposure, with binary variables for exposures to helminths, malaria and HIV infection (infected/not infected). Effects of helminth infection were then investigated in more detail by examining categories of infection intensity (none, light, moderate or heavy). For these analyses, cross-tabulations were made between selected risk factors and the presence of anaemia. Logistic regression was used to estimate unadjusted and adjusted odds ratios (OR). Likelihood ratio tests were used to determine P-values. Continuous variables for anaemia (Hb level) and infection intensity (egg or parasite count) were then used to examine effects of intensity in more detail among infected participants using linear regression. Adjustment was made for the same potential confounders in logistic and linear regression models.
3
Results
A total of 15 035 women registered at the antenatal clinic during the recruitment period, of whom 11 783 were assessed for inclusion in the study and 3163 were considered eligible and screened. The commonest reasons for ineligibility were residence outside the study area (6243), unwillingness to have an HIV test (1186), unwillingness to join the study (874) and enrolment during an earlier pregnancy (115). Of the 3163 screened, 2515 were enrolled; 8 of these were subsequently excluded because they had been enrolled during a previous pregnancy. Of the 648 women screened but not enrolled, the majority (596) failed to return for enrolment and only 15 brought a stool sample. Since intestinal helminth infection detected by stool analysis was a focus of interest, this analysis was confined to the 2507 women who were enrolled in the trial and for whom all or almost all relevant data were available.
3.1
Characteristics of the study women
Maternal age ranged from 14 years to 47 years (mean 23.6 years). The majority were Baganda (49.1%), the predominant tribe of the district. Most (83.8%) were married, with 13.4% single, 0.6% widows and 2.3% divorced or separated. Education varied from none (3.9%), to primary (50.5%), secondary (37.3%) and tertiary (8.4%) and most women were poor (85.1% with a personal income of less than £10 per month). Primigravidae comprised 27.7% of the women studied.
3.2
Prevalence of infections and anaemia
Complete data were obtained for Kato–Katz assays from 2498 women, for Strongyloides assays from 2485 women, for Mansonella from 2499 women and for malaria from 2459 women. The prevalence of hookworm was 44.5%, Mansonella perstans 21.3%, S. mansoni 18.3%, Strongyloides stercoralis 12.3%, T. trichiura 9.1%, Ascaris lumbricoides 2.3%, Trichostrongylus sp. 1.0%, Hymenolepis nana 0.2% and Loa loa <0.1%. Ova of Fasciola hepatica and Dicrocoelium dendriticum were found in samples from one and five women, respectively, but infection was not confirmed in follow-up samples and eggs probably originated from liver or offal in the women's diet; detection of ova of these two species was not considered to indicate infection. The prevalence of asymptomatic Plasmodium falciparum malaria parasitaemia was 10.9% and HIV infection 11.9%.
The prevalence of anaemia (Hb < 11.2 g/dl) was 39.7%. Six women were enrolled in error with Hb < 8 g/dl by both methods (Coulter analyser results 5.6–7.9 g/dl). These, as well as the 37 women enrolled prior to March 2005 with haemoglobinometer results above but Coulter analyser results below 8 g/dl, have been retained in this analysis.
3.3
Associations between characteristics of pregnant women and anaemia in pregnancy
Relationships between characteristics of the participating women and anaemia are shown in Table 1. The prevalence of anaemia declined with age. Anaemia was associated with tribe, with Basoga most likely to be anaemic. Anaemia showed no association with women's socioeconomic index, but low household socioeconomic status was associated with anaemia. Primigravidae were more likely to be anaemic than multigravidae.
3.4
Relationship between infections and anaemia in pregnancy
Relationships between infections and anaemia are shown in Table 2. None of the helminths showed an association with anaemia when considered using categorical variables for the presence or absence of infection. Hookworm showed a weak positive association, but this was reduced in the adjusted model.
Malaria parasitaemia and HIV infection were strongly associated with anaemia; for malaria the effect was unchanged and for HIV it increased after adjusting for potential confounding factors. Malaria was also associated with HIV infection (OR 1.81, 95% CI 1.30–2.53; P = 0.001); the association between HIV and anaemia was reduced slightly, but not explained, when malaria was added to the model (adjusted OR (AOR), adjusted for age, tribe, socioeconomic index, gravidity and malaria: 2.27, 95% CI 1.74–2.97; P < 0.001). The likelihood of being anaemic was particularly high among women with both HIV infection and malaria compared with women with neither infection (AOR 6.50, 95% CI 3.27–12.95; P < 0.001). Comparing those with HIV only to those with neither gave an AOR of 2.30 (95% CI 1.73–3.05; P < 0.001) and comparing those with malaria only to those with neither gave an AOR of 3.09 (95% CI 2.28–4.20; P < 0.001).
Hookworm and Mansonella showed positive associations with malaria parasitaemia, and hookworm showed a negative association with HIV infection, but adjusting for these infections had minimal effect on the observed associations between helminths and anaemia (data not shown).
Attributable fractions for anaemia were 3.1% for hookworm, 12.3% for malaria and 10.2% for HIV infection.
3.5
Relationship between anaemia and infection intensity
Associations between infections and anaemia were further explored by examining infection intensity (Table 3). The prevalence of anaemia increased with each category of hookworm infection intensity. However, this trend was greatly weakened, showing no evidence of association after adjusting for age, tribe, socioeconomic index, gravidity, malaria and HIV. On the other hand, there was a small negative association between log10 hookworm egg count and Hb in hookworm-infected women (adjusted regression coefficient −0.23, 95% CI −0.38 to −0.09; P = 0.002), i.e. women with the highest hookworm intensity (approximately 10 000 epg) had, on average, a Hb level 0.69 g/dl lower than those with the lowest detectable egg counts (12 epg). Anaemia prevalence was higher in women with heavy S. mansoni infection than in those with lower intensity infection or no infection, but the number of such women was small and the effect was not statistically significant. Infections with Trichuris were light in all but six of the infected women, therefore associations between anaemia and moderate-to-heavy Trichuris infections could not be examined. There was no evidence of an association between Hb and egg count for women infected with S. mansoni or Trichuris.
There was a negative association between log10 malaria parasite count (parasites per 200 white blood cells) and Hb, but this was reduced in the adjusted model (crude regression coefficient −0.27, 95% CI −0.53 to −0.01, P = 0.039; adjusted regression coefficient −0.17, 95% CI −0.45 to 0.11, P = 0.206). Hb was not significantly associated with log10 CD4+ T-cell count among HIV-positive women (adjusted regression coefficient 0.51, 95% CI −0.17 to 1.19; P = 0.120).
3.6
Anaemia in women excluded from enrolment
Hb < 8 g/dl was an exclusion criterion. Among the 648 women who were screened but never enrolled, Hb < 8 g/dl was the reason given in 17 cases, but Coulter analyser Hb was <8 g/dl for a further 37 women excluded for other reasons. The prevalence of anaemia was 281/648 (43.4%) among those excluded compared with 996/2507 (39.7%) among those enrolled (P = 0.093), giving an overall prevalence of 40.5% among all women screened. Since stool results were only available for 15 of the excluded women, associations with intestinal helminths could not be analysed. Of the 54 women excluded and with Coulter Hb < 8 g/dl, two had stool results: both had hookworm infections (one light and one moderate intensity) and neither had schistosomiasis. A sensitivity analysis was performed assuming that all women excluded and with Coulter Hb < 8 g/dl had hookworm. As expected, a slightly stronger association was obtained, but this was again reduced after adjusting for confounding factors (crude OR 1.29, 95% CI 1.10–1.51, P = 0.001; AOR adjusted for age, tribe, socioeconomic indices and gravidity 1.19, 95% CI 1.00–1.41, P = 0.047).
Associations with malaria and HIV were similar in the excluded group to those in the enrolled group, with crude ORs of 3.73 (95% CI 2.23–6.23; P < 0.001) and 1.57 (95% CI 1.06–2.34; P = 0.026), respectively.
4
Discussion
This study suggests that, among pregnant women in Entebbe, Uganda, malaria and HIV are more important infectious causes of anaemia than helminths. No association was observed between mild-to-moderate anaemia and any species of helminth, and a weak association between anaemia and increasing intensity of hookworm infection was reduced after adjusting for confounding factors. Anaemia was slightly more common among women heavily infected with S. mansoni, but the number of such women was small.
In keeping with the exclusion of women with Hb < 8 g/dl, excluded women were more likely to be anaemic than enrolled women, but this had minimal impact on the overall estimate of prevalence of anaemia (40.5%). Given the recognised ability of hookworm to cause anaemia (Bondevik et al., 2000; Hotez et al., 2004; Shulman et al., 1996), we conducted a sensitivity analysis to estimate the effect of hookworm if all women with Hb < 8 g/dl and no stool result had hookworm. The result was in keeping with a possible effect of hookworm, but after adjusting for confounding factors the effect was small. Associations between anaemia and malaria and between anaemia and HIV infection were similar in enrolled and excluded women.
Our investigations for intestinal helminths used only one stool sample from each woman, meaning that a proportion of women with low-intensity infections will have been misclassified as uninfected and that estimates of intensity will have been imprecise (Hall, 1981; Utzinger et al., 2001). Recent comparable studies have the same limitation (Ajanga et al., 2006; Bondevik et al., 2000; Dreyfuss et al., 2000; Larocque et al., 2005). In our study this may, again, be important for hookworm, where there was a small increase in anaemia with infection intensity, but may not explain the lack of association for schistosomiasis where there was no suggestion of an effect of light-to-moderate infections. Our method of investigation for Mansonella and intensity does not suffer from this limitation: repeat examinations among 1971 women showed 96% agreement for the binary variable (infected/uninfected), with an ICC for mf/ml of 0.87 (95% CI 0.85–0.88) (unpublished data).
Not all pregnant women in Entebbe attend the district hospital antenatal clinic, but a community survey undertaken in the study area showed an increase in the proportion choosing this clinic during the recruitment period, to approximately 80%, and most of the personal and socioeconomic characteristics of women choosing, or not choosing, to attend the district hospital clinic were similar (unpublished data). Thus, our results are likely to be reasonably representative of pregnant women in this area.
Recent studies from different settings give similar results in relation to the effects of hookworm and S. mansoni on anaemia in pregnancy. In Peru (where hookworm and Trichuris are predominant), in Tanzania (S. mansoni and hookworm), in the Democratic Republic of Congo (Ascaris and hookworm) and in Java (Trichuris and hookworm), no association was observed between anaemia and infection with any single species (Ajanga et al., 2006; Kalenga et al., 2003; Larocque et al., 2005; Nurdia et al., 2001). In Peru there was an association between anaemia and higher hookworm intensities, and in Java there was a negative association between serum ferritin and hookworm, suggesting an effect of hookworm on iron status but not anaemia; in Tanzania, in an area of higher S. mansoni prevalence and intensity, a strong association between anaemia and heavy S. mansoni infection was observed. Infection intensity thus appears to be important, with light-to-moderate hookworm or S. mansoni infections having relatively weak effects on Hb levels. However, a second important factor is the underlying nutritional status of the women. In two studies in Nepal, among populations perhaps poorer and less well nourished than ours (as indicated by items owned, anthropometry and vitamin A status (Dreyfuss et al., 2000) and with a traditionally vegetarian diet (Bondevik et al., 2000)), anaemia showed a significant association with hookworm infection (Bondevik et al., 2000; Dreyfuss et al., 2000). The effects of hookworm infection are partially mediated by iron deficiency (Bondevik et al., 2000; Olsen et al., 1998) and in the trial conducted in Sierra Leone iron-folate supplements had a greater benefit for anaemia in pregnancy than treatment with albendazole (Torlesse and Hodges, 2001).
We found no suggestion of an association between anaemia and any other helminth species that was common in our environment (Mansonella, Trichuris or Strongyloides). This again is in agreement with other studies (Dreyfuss et al., 2000; Larocque et al., 2005; Nurdia et al., 2001). Larocque et al. (2005) noted a stronger effect of moderate-to-heavy hookworm infection when combined with moderate-to-heavy Trichuris infection. Although there was broad overlap between the confidence intervals for these effects in their analysis, such an effect is plausible, given evidence in children that heavy Trichuris infections (>10 000 epg) can be associated with anaemia (Ramdath et al., 1995). In our study, as in that reported by Nurdia et al. (2001), no such heavy Trichuris infections were observed.
The strong effects of malaria and HIV contrast with the weak effect of hookworm and the lack of effects of other helminths in this study.
Of note, women were screened for this study when they were apparently healthy, thus both malaria and HIV infection were largely asymptomatic. The effects of these infections are not mediated by iron deficiency and may override any benefit of good nutrition. The importance of malaria as a cause of anaemia in pregnancy is well established (Shulman and Dorman, 2003). HIV infection is also a recognised cause of anaemia (Belperio and Rhew, 2004), and anaemia may be one mechanism by which it causes adverse birth outcomes (Dairo et al., 2005; McIntyre, 2003).
There are compelling reasons for preventing and treating malaria and HIV during pregnancy (Shulman and Dorman, 2003; ter Kuile et al., 2004). Our results highlight that anaemia is among them. On the other hand, our results as well as recent literature suggest that associations between helminth infections and anaemia in pregnancy are weaker, with regional variations that may be based on nutrition and intensity of helminth infection. These findings are relevant when estimating the relative disease burden of helminths and other infections and the relative value of possible interventions in pregnancy. Globally, the majority of helminth infections are of low intensity so, in some settings, the benefit of routine deworming during pregnancy in relation to anaemia may be modest. The effects of deworming during pregnancy on other parameters, including birth outcome, birth weight and long-term effects on health in infancy and childhood, also need to be considered (Christian et al., 2004). The forthcoming results of our ongoing trial of deworming in pregnancy are expected to contribute further to this debate.
Authors’ contributions
AME designed the study; JN and CA carried out interviews and recruited participants; MO and HM carried out clinical assessments; NO and DK carried out laboratory assessments; LM, PW and LAM analysed and interpreted the data; LM, PW, LAM and AME drafted the manuscript. All authors reviewed and approved the final manuscript. LM and AME are guarantors of the paper.
Funding
Wellcome Trust Career Post fellowship held by Dr Elliott, grant number 064693; PMTCT programme, Ministry of Health, Uganda.
Conflicts of interest
None declared.
Ethical approval
The Science and Ethics Committee, Uganda Virus Research Institute, the Uganda National Council for Science & Technology, and the London School of Hygiene & Tropical Medicine.

anaemia pregnancy helminth malaria hiv uganda


A multidisciplinary, multifactorial intervention program reduces postoperative falls and injuries after femoral neck fracture

Introduction This study evaluates whether a postoperative multidisciplinary, intervention program, including systematic assessment and treatment of fall risk factors, active prevention, detection, and treatment of postoperative complications, could reduce inpatient falls and fall-related injuries after a femoral neck fracture.
Introduction
Nearly all hip fractures occur as a result of a fall [1] and many fall again soon after sustaining the fracture [2]. Osteoporosis with low bone mineral density (BMD) puts older people who fall at high risk of sustaining fractures [3, 4]. A first hip fracture is associated with a 2.5-fold increased risk of a subsequent fracture [5]. A population-based study among people aged 85 years or older showed that 21% of those with a hip fracture had suffered at least two hip fractures [6].
Previous research has identified several fall risk factors such as comorbidity, functional disability, previous falls, and use of drugs [7–11] but also aging [12, 13] and among the oldest old, male sex [14]. Delirium, which is very common after hip fracture surgery, especially among those with a cognitive decline [15, 16], has been found to be one of the most important risk factors for falls among older people [10].
Multi factorial intervention strategies among community-living older people can prevent falls [17–20] and are the recommendations in fall prevention interventions nowadays [21]. The recommendations in fall prevention programs is that they should include gait training, advice on use of assistive devices, medication reviews, exercise programs including balance training, treatment of hypotension, environmental modification, and treatment of cardiovascular disorders. In long-term care the program recommends also to include staff education [21]. Most fall prevention studies are performed in the community, but multidisciplinary and multifactorial interventions have also been shown to be beneficial in residential care facilities [22].
Few fall prevention studies have been carried out in hospitals; there have been a few studies with single interventions among older patients in rehabilitation units, without any significant effects [23–25]. Recently two studies, one using multiple interventions [26] and one using a multidisciplinary fall prevention approach [27], have demonstrated a reduction in falls. None of these fall prevention studies have focused on hip fracture patients or tried to reduce postoperative complications as a fall prevention measure.
Considering the lack of fall prevention studies in hospitals, especially after recent hip fracture surgery, this is an area of interest for study. The aim of this study was thus to evaluate if a postoperative multidisciplinary, multifactorial intervention program could reduce inpatient falls and fall-related injuries in patients with femoral neck fractures.
Methods
Recruitment and randomization
This study included patients with femoral neck fracture aged ≥70 years, consecutively admitted to the orthopedic department at the Umeå University Hospital, Sweden, between May 2000 and December 2002, and the study was designed according to the CONSORT guidelines [28].
In Sweden different surgery methods are used depending on the displacement of the femoral neck fracture. In the present study patients with undisplaced fracture were operated on using internal fixation (IF) and patients with displaced fracture were operated on using hemiarthroplasty (HAP). If patients had severe rheumatoid arthritis, severe hip osteoarthritis, or pathological fracture they were excluded, by the surgeon on duty, because of the need for a different surgery method, such as total hip arthroplasty (THA). Patients with severe renal failure were excluded, by the anesthesiologist, because of their morbidity. Patients being bedridden before the fracture occurred were also excluded.
In the emergency room the patients were asked both in writing and orally if they were willing to participate in the study. The next of kin was always asked prior to the inclusion in patients with cognitive impairment. The patients or their next of kin could at any time decline participation. A total of 258 patients met the inclusion criteria; 11 patients declined to participate and 48 patients were not invited to participate because they had sustained the fracture in the hospital or the inclusion routines failed (Fig. 1). These 59 patients were more likely to be men (p = 0.033) and living in their own house/apartment (p = 0.009), but there was no difference in age (p=0.354) compared to the participating patients. The remaining 199 patients (Table 1) consented to participate. All patients received the same preoperative treatment.
Patients were randomized, to postoperative care in a geriatric ward with a special intervention program or to conventional care in an orthopedic ward, in opaque sealed envelopes. The lots in the envelopes were sequentially numbered. All participants received this envelope while in the emergency room but the envelope was not opened until immediately before surgery to ensure that all patients received similar preoperative treatment. Persons not involved in the study performed these procedures.
The randomization was stratified according to the operation methods used in the study. Depending on the degree of dislocation, the patients were treated with IF using two hook-pins (Swemac Ortopedica, Linköping, Sweden) (n=38 intervention vs n = 31 control) or with bipolar hemiarthroplasty (Link, Hamburg, Germany) (n = 57 vs 54). Basocervical fractures (n = 7 vs 10) were operated on using a dynamic hip screw (DHS, Stratec Medical, Oberdorf, Switzerland) and one had a resection of the femoral head due to a deterioration in medical status and one died before surgery (both were in the control group).
Fig. 1Flow chart for the randomized trialTable 1Basic characteristics and assessments during hospitalization among participants in the intervention and control groups. SD standard deviation, ADL activity of daily living Intervention (n = 102)Control (n = 97)p valueSociodemographic Age, mean±SD82.3 ± 6.682.0 ± 5.90.724 Females74740.546 Independent living before the fracture66600.677Health and medical problems Stroke (n = 102/93)29200.265 Dementia28360.145 Previous hip fracture (n = 102/96)a16140.829 Depression (n = 102/95)33450.031 Diabetes (n = 102/95)23170.417 Cardiovascular disease (n = 101/93)57530.938Medications on admission Number of drugs, mean±SD5.8 ± 3.85.9 ± 3.60.867 Antidepressants 29450.009Sensory impairments Impaired hearing (n = 94/82)42340.667 Impaired vision (n = 91/74)37270.584Functional performance before fracture Use of roller walker (n = 101/93)56520.948 Use of wheelchair (n = 101/93)23160.334 Previous falls, last month (n = 99/90)b24250.580 Walking independently, at least indoors (n = 101/94)85850.191 Staircase of ADL, median (Q1,Q3) (n = 92/88)5 (1–7.75)5 (0.25–7)0.859Assessments during hospitalization Mini Mental State Examination, mean±SD (n = 93/90)17.4 ± 8.215.7 ± 9.10.191 Organic Brain Syndrome Scale, mean±SD (n = 94/90)10.1 ± 10.812.5 ± 11.40.148 Geriatric Depression Scale, mean±SD (n = 81/68)5.2 ± 3.64.5 ± 3.50.271aExcept for the present hip fracturebExcept for the fall that caused the hip fracture
Intervention
The intervention ward was a geriatric unit specializing in geriatric orthopedic patients. The staff worked in teams to apply comprehensive geriatric assessments, management, and rehabilitation [29, 30]. Active prevention, detection, and treatment of postoperative complications such as falls, delirium, pain, and decubitus ulcers was systematically implemented daily during the hospitalization (Table 2). The staffing at the intervention ward were 1.07 nurses/aides per bed.
Table 2Main content of the postoperative program and differences between the two groups Intervention groupControl groupWard layoutSingle and double roomsSingle, double, and four-bed rooms24-bed ward, extra beds when needed27-bed ward, extra beds when neededThe geriatric control ward was similar to the intervention wardStaffing1.07 nurses/aides per bed1.01 nurses per bedTwo full-time physiotherapistsTwo full-time physiotherapistsTwo full-time occupational therapists0.5 occupational therapist0.2 dieticianNo dieticianThe geriatric control ward had staffing similar to the intervention wardStaff educationA 4-day course in caring, rehabilitation, teamwork, and medical knowledge including sessions about how to prevent, detect, and treat various postoperative complications such as postoperative delirium and fallsNo specific education before or during the projectTeamworkTeam included registered nurses (RN), licensed practical nurses (LPN), physiotherapists (PT), occupational therapists (OT), dietician, and geriatriciansNo corresponding teamwork at the orthopedic unitClose cooperation between orthopedic surgeons and geriatricians in the medical care of the patientsThe geriatric ward, where some of the control group patients were cared for, used teamwork similar to that in the intervention wardIndividual care planningAll team members assessed each patient as soon as possible, usually within 24 h, to be able to start the individual care planningIndividual care planning was used in the orthopedic unit but not routinely as in the intervention wardTeam planning of the patients’ individual rehabilitation process and goals twice a weekAt the geriatric rehabilitation unit there was weekly individual care planningPrevention and treatment of complicationsInvestigation as far as possible regarding how and why they sustained the hip fracture, through analyzing external and internal fall risk factorsNo routine analysis of why the patients had fractured their hipsAn action to prevent new falls and fractures was implemented including global ratings of the patients’ fall risk every week during team meetingsNo attempt was made to systematically prevent further fallsCalcium and vitamin D and other pharmacological treatments for osteoporosis were used when indicatedNo routine prescription of calcium and vitamin DActive prevention, detection, and treatment of postoperative complications such as delirium, pain, and decubitus ulcers was systematicAssessments for postoperative complications were made with check-ups for, i.e., saturation, hemoglobin, nutrition, bladder and bowel function, home situation etc., but these check-ups were not carried out systematically as in the intervention groupOxygen-enriched air during the 1st postoperative day and longer if necessary until the measured oxygen saturation was stableUrinary tract infections and other infections were screened for and treatedIf a urinary catheter was used it should be discontinued within 24 h postoperativelyRegular screening for urinary retention, and prevention and treatment of constipationBlood transfusion was prescribed if B-hemoglobin  <100 g/l and  <110 for those at risk of delirium or those already deliriousIf the patient slept badly, the reason was investigated and the aim was then to treat the causeNutritionFood and liquid registration was systematically performed and protein-enriched meals were served to all patients during the first 4 postoperative days and longer if necessaryA dietician was not available at the orthopedic unitNutritional and protein drinks were served every dayNo routine nutrition registration or protein-enriched meals were available for the patientsRehabilitationMobilization within the first 24 h after surgeryMobilization usually within the first 24 hThe training included both specific exercise and other rehabilitation procedures delivered by a PT and OT, as well as basic daily ADL performance training, by caring staff. The patients should always do as much as they could by themselves before they were helpedThe PT on the ward mobilized the patients together with the caring staff. The PT aimed to meet the lucid patients every day. Functional retraining in ADL situations was not always given. The OT at the orthopedic unit only met the patients for consultationThe rehabilitation was based on functional retraining with special focus on fall risk factorsThe geriatric control ward had both specific exercise and other rehabilitation procedures delivered by a PT and OT, similar to the intervention ward but did not systematically focus on fall risk factorsHome visit by an OT and/or a PTNo home visits were made by staff from the orthopedic unit
The control ward was a specialist orthopedic unit following the conventional postoperative routines. A geriatric unit, specializing in general geriatric patients, was used for those who needed longer rehabilitation (n = 40). The staffing at the orthopedic unit was 1.01 nurses/aides per bed and 1.07 for the geriatric control ward. The main content of both the intervention program and the conventional care is described in Table 2.
The staffs on the intervention and control wards were not aware of the nature of the present study.
Data collection
Two registered nurses were employed and performed the assessments during hospitalization.
Medical, social, and functional data were collected from the patients, relatives, staff, and medical records on admission. Complications during hospitalization, including falls, length of stay, morbidity, and mortality, were systematically registered in the medical and nursing records. Nurses are obliged by law to document any falls in the records [31]. A fall was defined as an incident when the patient unintentionally came to rest on the floor and included syncopal falls. Numbers of falls and time lapse to first fall after admission were calculated. The Abbreviated Injury Scale (AIS) [32] was used to classify the injuries resulting from a fall. The maximum injury (MAIS) connected with each incident was recorded.
A few days after surgery, patients were assessed and interviewed regarding their cognitive status using the Mini Mental State Examination (MMSE) [33]. The modified Organic Brain Syndrome Scale (OBS Scale) [34] was used to assess cognitive, perceptual, emotional, and personality characteristics as well as fluctuations in clinical states. Mental state changes were also documented from medical records. Depression during hospitalization was diagnosed due to current treatment with antidepressants and depression screened using the Geriatric Depression Scale (GDS-15) [35] in combination with depressive symptoms observed and registered by the OBS Scale. The patients’ vision and hearing were assessed by their ability to read 3-mm block letters with or without glasses, and their ability to hear a normal speaking voice from a distance of 1 m. Activities of daily living (ADL) prior to the fracture were measured retrospectively using the Staircase of ADL [36].
A geriatrician, unaware of study group allocation, analyzed all assessments and documentation, after the study was finished, for completion of the final diagnoses according to the same criteria for all patients.
The Ethics Committee of the Faculty of Medicine at Umeå University approved the study (§ 00-137).
Statistical analysis
The sample size was calculated to detect a 50% reduction of number of fallers between the intervention and control groups at a significance level of 0.050, based on our previous multifactorial fall intervention study in institutional care [22]. Student’s t-test, Pearson’s χ2 test, and the Mann-Whitney U test were performed to analyze group differences regarding basic characteristics and postoperative complications.
We analyzed outcomes on an intention to treat basis. The incidence of falls between intervention and control groups was compared in three ways. First, an unadjusted comparison using Pearson’s χ2 and Fisher’s exact test regarding number of patients who fell and injuries. Second, the fall incidence rate was compared between intervention and control groups by calculating the fall incidence rate ratio (IRR) using a negative binomial regression, with adjustment for observation time and for overdispersion. Negative binomial regression (Nbreg) is a generalization of the Poisson regression model and is recommended for evaluating the efficacy of fall prevention programs [37]. Third, a Cox regression was used to compare the time lapse to first fall between groups (hazard rate ratio, HRR). The difference in fall risk between groups was further illustrated by a Kaplan-Meier graph.
Basic characteristics that differed between the intervention and the control groups, corresponding to a p value  <0.150 (depression, antidepressants, and dementia, Table 1), were considered as covariates in the Poisson (Nbreg) and the Cox regression models. However, the inclusion of these variables had only marginal effects on the log-likelihood values of the models as well as on the IRR and HRR values and standard errors for the group allocation variable (intervention or control). In addition, none of the variables showed significant effects on the dependent variable and are therefore not included in the Poisson (Nbreg) and Cox regression analyses.
Pearson’s χ2 test and Fisher’s exact test were also used to analyze the associations between falls and days with delirium between the groups.
All calculations were carried out using SPSS v 11.0 and STATA 9 statistical software for Macintosh. A p value  <0.050 was considered statistically significant.
Results
During hospitalization 12 patients in the intervention group sustained 18 falls (range: 1–3) and in the control group 26 patients sustained 60 falls (30 falls in the orthopedic unit and 30 in the geriatric control unit) (range: 1–11). Among patients with dementia 1 patient sustained a single fall in the intervention group and 11 patients sustained 34 falls in the control group (Table 3).
Table 3Falls during hospitalization. CI confidence interval, IRR incidence rate ratio Intervention (n = 102)Control (n = 97)p valueNumber of falls1860Postoperative in-hospital days2,8603,685Crude fall incidence rate (number of falls/1,000 days)6.2916.28IRR (95% CI)0.38 (0.20–0.76)a1.00 (Ref.)0.006Number of fallers12260.007Number of fallers with injuries due to falls3150.002Number of fallers with fractures due to falls040.055Number of falls among people with dementia134IRR (95% CI) among people with dementia0.07 (0.01–0.57)a1.00 (Ref.)0.013Number of fallers among people with dementia (n = 28/36)1110.006aNegative binomial regression analyses adjusted for overdispersion and controlled for dementia, depression, and use of antidepressants
The crude postoperative fall incidence rate was 6.29/1,000 days in the intervention group vs 16.28/1,000 days in the control group. Using a negative binomial regression, the fall incidence was significantly lower in the intervention group, IRR 0.38 (95% CI: 0.20–0.76, p = 0.006), and among patients with dementia, IRR 0.07 (95% CI: 0.01–0.57, p = 0.013) (Table 3). In Fig. 2, a Kaplan-Meier survival analysis of time lapse to first fall illustrates the difference between the two groups with a significantly reduced fall rate in the intervention group (log rank p value 0.008).
Fig. 2Kaplan-Meir survival graph
The difference in fall risk, expressed as time lapse to first fall, was compared between intervention and control groups in a Cox regression (HRR). Including all patients in the calculation, the fall risk was significantly lower in the intervention group, HRR 0.41 (95% CI: 0.20–0.82, p = 0.012).
There were in total 3 minor or moderate injuries (MAIS 1-2) in the intervention group compared to 15 in the control group according to the AIS. The serious injuries (MAIS 3) were new fractures of which four, two hip fractures, one rib fracture with pneumothorax, and one with multiple skull fractures, occurred in the control group and none in the intervention group (Fisher’s exact test: p = 0.055).
Three of the patients who fell in the intervention group (25%) and 12 in the control group (46%) fell during a day when they were delirious (p = 0.294). Analyzing the number of falls revealed that 4 of 18 (22%) falls in the intervention group and 27 of 60 (45%) in the control group occurred on a day when the patient was delirious, p = 0.083.
Apart from the falls there were fewer other postoperative complications in the intervention group, such as fewer patients with postoperative delirium (p=0.003) and fewer delirious days (p ≤ 0.001), urinary tract infections (p = 0.005), sleeping disturbances (p = 0.009), nutritional problems (p = 0.038), and decubitus ulcers (p = 0.010). The postoperative in-hospital stay was shorter in the intervention group, 28.0 ± 17.9 days vs 38.0 ± 40.6 days, p = 0.028. Among those ten with the longest postoperative in-hospital stays in the control group there were eight patients with any fall and two had had new fractures.
Discussion
The present study shows that the number of falls and time lapse to first fall can be reduced during in-hospital rehabilitation after a femoral neck fracture. A multidisciplinary, multifactorial geriatric care program with systematic assessment and treatment of fall risk factors as well as active prevention, detection, and treatment of other postoperative complications resulted in fewer patients who fell, a lower total number of falls, and fewer injuries.
To our knowledge this is the first fall intervention study in this group of patients, despite the fact that this is a group of patients with a high fall risk. In general there are few fall prevention studies in hospital settings. Two [26, 27] with positive outcomes in other patient groups and on subacute wards have recently been published. The first one [26] reduced falls at three subacute rehabilitation wards, but the differences were most obvious after 45 days of observation. Thus the results were not comparable with those from the present study, which included both the acute and rehabilitation hospital stay. The other study [27] resulted in fewer fallers, falls, and injuries on a geriatric ward but the differences disappeared when the results were adjusted for observation time. Those studies used a multidisciplinary approach in their fall intervention similar to that used in the present study, but in the present study we have, in addition, focused on inpatient complications associated with falls such as delirium and urinary tract infections. One of those studies [27] tried to manage the delirious patients using bedrails, alarms, and changing the furniture arrangements for the patients, but no mention was made of any prevention and treatment of the underlying causes of delirium. The use of physical restraints was not included in the intervention program in the present study. The studies above used fall risk assessment tools to recognize those with a high fall risk. In the present study, we used a rehabilitation and care program including assessment of risk factors for falls and global ratings for each patient during team meetings. A critique of fall risk assessment tools is that few have been tested for validity and reliability testing in a new independent sample. When using fall prediction tools in different clinical settings the specificity decreases [38].
A limitation in the present study is that some falls could have been missed, but we presume that there were very few. For one thing the nurses are obliged to document falls in the records. Also hip fracture surgery patients can hardly get up by themselves after a fall so soon after the surgery and are, therefore, bound to be noticed; but if there were any missing falls there would probably be no difference between the groups. Another limitation is that the fall registration could not be blinded regarding group allocation, but the staffs on each ward were not aware of the comparison with another ward regarding falls and injuries. The study sample is also quite small, but the sample size is calculated according to the results from a previous study [22]. The method of concealment could have been improved, but one strength was that none from the research team performed this procedure and the envelopes were not opened until the intervention was to begin. Other strengths were the intention to treat analyses, the few patients who refused to participate, and that there were no crossover effects due to staff changing wards during the study period.
One may speculate that the successful reduction in number of falls in the present study could be a result of the active prevention, detection, and treatment of postoperative complications after surgery. During the period of hospitalization there were differences between the groups regarding some complications associated with falls among older people in residential care facilities and in hospitals, such as delirium and urinary tract infections. The reduction of postoperative delirium can probably explain much of the difference between the groups regarding the numbers of falls and the number of patients who fell. There are studies that have found that delirium is an important risk factor for falls [10]. Demented patients especially are at high risk of developing delirium when they are treated for femoral neck fractures [15, 16] and these patients seemed to have benefited most in this study from the intervention program regarding prevention of postoperative falls. Our findings support an earlier non-randomized study that fewer injurious falls occur when the incidence and duration of delirium was reduced [39].
The investigation into why the patients had fractured their hip and why they fell may also have influenced the result, as well as the investigation and rehabilitation concerning external fall risk factors such as the use of walking aids, safe transfers, balance, and mobility. It seems that teamwork and individual care planning alone do not have the same effect on falls, as half the falls in the control group occurred in the geriatric control ward, a ward specializing in geriatric patients where teamwork, as well as individual care planning, is applied.
In the community and residential care facilities, interdisciplinary and multifactorial fall prevention studies have shown positive effects on the reduction in the number of falls and injuries [19, 22]. Among those with cognitive decline or dementia there is no evidence that such strategies prevent falls [40, 41], but the present study allowed the conclusion that at least during the in-hospital stay, this group of patients could benefit from such strategies. The reduced number of falls and injuries also probably contributed to the shorter hospitalization seen in the intervention group. The program seems easy applicable both in the acute postoperative care as well in the post-acute rehabilitation settings and except for the staff education there were no increased costs.
Conclusion
A team applying comprehensive geriatric assessment and rehabilitation, including prevention, detection, and treatment of fall risk factors, can successfully prevent inpatient falls and injuries, even in patients with dementia.

intervention
hip fracture
in-hospital
accidental falls
elderly


Capillary-assembled microchip as an on-line deproteinization device for capillary electrophoresis

A capillary-assembled microchip (CAs-CHIP), prepared by simply embedding square capillaries in a lattice polydimethylsiloxane (PDMS) channel plate with the same channel dimensions as the outer dimensions of the square capillaries, has been used as a diffusion-based pretreatment attachment in capillary electrophoresis (CE). Because the CAs-CHIPs employ square-section channels, diffusion-based separation of small molecules from sample solutions containing proteins is possible by using the multilayer flow formed in the square section channel. When a solution containing high-molecular-weight and low-molecular-weight species makes contact with a buffer solution, the low-molecular-weight species, which have larger diffusion coefficients than the high-molecular-weight species, can be collected in a buffer-solution phase. The collected solution containing the low-molecular-weight species is introduced into the separation capillary to be analyzed by CE. This type of system can be used for CE analysis in which pretreatment is required to remove proteins. In this work a fluorescently labeled protein and rhodamine-based molecules were chosen as model species and a feasibility study was performed.
Introduction
On-line combination of sample pretreatment and electrophoretic separations, for example capillary electrophoresis (CE) and microchip-based capillary electrophoresis (MCE), has been focused on by many researchers, because of the possibilities of integrating complicated pretreatment procedures. During the past decade a variety of approaches have been used to combine pretreatment with CE separations. The “single capillary approach”, involving position-selective immobilization of functional molecules or polymers inside a capillary, has been used to demonstrate preconcentration or enzymatic reaction before CE separation [1, 2], and in-capillary preconcentration during the electrophoretic process [3–8] has provided a simple system enabling direct application of the capillary in commercial equipment. The “microchip-based approach”, recently reviewed by many authors [9–11], has enabled integration of a variety of complicated pretreatment processes with the separation channel on a single microchip. These approaches have different advantageous features for each purpose. From the standpoint of total system design, however, a “combination approach” involving the use of a flow-system or a functionalized capillary together with a separation capillary provides a more flexible system designed for particular analyte. Ye et al. reported connection of a trypsin-immobilized capillary with a separation capillary, by means of a home-built chip prepared by lamination of Lexan and Parafilm, and demonstrated on-line digestion and CE separation [12]. Kuban et al. and Fang et al. reported “flow-injection capillary electrophoresis (FI-CE)” comprising a flow-injection system and a separation capillary [13, 14]. This system enabled various pretreatment processes to be combined with CE separation and has since been expanded to the use of MCE to achieve rapid separations. These systems have been reviewed by Chen et al. [15]. Vizioli et al. prepared a monolithic polymer capillary, for selection of histidine-containing peptides, connected it to a CE separation capillary [16]. Fan et al. recently reported combination of an FI-CE system with in-capillary preconcentration using a dynamic pH junction [17].
In all the examples mentioned above, combination of a flow-reaction system or chemically functionalized conduits with a separation capillary played an important role, and would provide an attractive integrated device enabling flexible design for combining pretreatment and CE separation. In contrast, we have recently reported a new concept for fabricating a chemically functionalized microchip, called a “capillary-assembled microchip (CAs-CHIP)” [18]. The microchip is fabricated simply by embedding chemically functionalized square capillaries in a lattice PDMS channel chip which has the same channel dimensions as the outer dimensions of the square capillaries. By using this technique, we have constructed a multi-ion sensing system based on combination of several different ion-sensing capillaries and a valving and sensing system based on thermo-responsive polymer-immobilized capillaries and an enzyme-immobilized capillary [19, 20]. Because the CAs-CHIP enabled easy connection of different functional capillaries, we focused on this device as an on-line sample-pretreatment attachment for CE separation capillaries in a manner similar to the previously reported FI-CE system. In this instance the CAs-CHIP, prepared from a PDMS chip, served both as the device for pretreatment of the sample solution, by use of a variety of chemical processes, and the injection device. Interfacing the flow system and the separation capillary using a PDMS chip has already been reported by Bergstrom et al. [21, 22]. Because the CAs-CHIP employs a square-shaped channel structure, however, the characteristic multilayer flow formed in the square section channel can also be used as a useful pretreatment process which can be integrated. The CAs-CHIP is, therefore, expected to enable the integration of universal pretreatment processes by using different kinds of chemically-functionalized capillary and multilayer flow-based chemical processes; this is usually difficult to achieve using other reported techniques.
In this paper, we report preliminary results concerning the preparation of a CAs-CHIP as a deproteinization attachment for CE separation. Deproteinization was achieved by use of the multilayer flow obtained in the PDMS microchannel, and the small molecules separated from the mixed protein sample were injected into the separation capillary connected directly to the CAs-CHIP, to be analyzed by CE. In this work a fluorescently labeled protein and rhodamine-based molecules were chosen as model species, and feasibility study was performed.
Experimental
Square capillaries and reagents
Square capillaries of 300 μm outer width (flat-to-flat) and 50 μm inner width were purchased from Polymicro (Phoenix, AZ, USA). Before use of these capillaries the polyimide coating was removed by heating. Sylgard 184 silicone elastomer was purchased from Dow Corning (Midland, MI, USA). Reagents of the highest grade commercially available were used to prepare the aqueous solutions. Rhodamine B (RB), sulforhodamine (SR), and bovine serum albumin fluorescein conjugate (F-BSA) were purchased from Sigma (St Louis, MO, USA). All reagents were used without further purification. Distilled and deionized water had resistivity greater than 1.7 × 107 Ω cm−1 at 25°C.
Fabrication of CAs-CHIP-CE system by embedding square capillaries on a PDMS plate and bonding of a PDMS cover
Figure 1 shows the design of a CAs-CHIP-CE system. The PDMS plate for pretreatment was connected to the inlet of the separation capillary (left side of Fig. 1) and another PDMS plate was used to fix the detection position (right side of Fig. 1). Square capillaries were cut into appropriate lengths and embedded in the lattice microchannel network fabricated on the PDMS plate. The general procedure for fabrication of a lattice microchannel on a PDMS plate has been reported elsewhere [18]. Briefly, a glass mold with a lattice structure was prepared by cutting a 300-μm depth with 1 mm pitch, using a dicing saw with an edge 300 μm wide. The conventional PDMS molding process using the glass mold was then performed to prepare a PDMS mold. The second molding process using this PDMS mold gave the lattice microchannel network on the second PDMS plate. Plugged capillaries were prepared by introduction of PDMS prepolymer into the square capillaries (inner width 50 μm) and cured at 70°C for more than 5 h. These plugged capillaries were also cut and used to prepare the designed channel network. After embedding all the capillaries, a PDMS cover was bonded on top. For this, a spin-coated PDMS prepolymer on an acrylic plate (ca. 2 mm thick) was used as the cover plate, to fill the voids formed between the cover plate and the capillary-embedded PDMS plate [18]. PDMS prepolymer was spin-coated on the acrylic plate at 7000 rpm, then attached to the capillary-embedded PDMS plate before curing. Bonding was carried out by curing at 60°C for 12 h. Figure 2 shows the final CAs-CHIP-CE system with a 12-cm separation capillary. Depending on the resolution required, 12-cm (effective length 10 cm) and 48-cm (effective length 33 cm) separation capillaries were used. To apply the high voltage, two plastic vials, as solution reservoirs, were connected at the outlets of the PDMS channel and the separation capillary.
Fig. 1General concept for fabricating a diffusion-based pretreatment–CE separation system using a capillary-assembled microchip (CAs-CHIP). The plugged capillaries indicated as gray parts are actually square capillaries with 50 μm square-shaped conduits blocked with PDMS. In this figure, for simplicity, these conduits are not shownFig. 2Photograph of a fabricated CAs-CHIP-CE device
Operating procedures
Channels (or capillaries) on the CAs-CHIP-CE system were washed sequentially with methanol, 0.1 mol L−1 NaOH, 0.1 mol L−1 HCl, and water, then rinsed and preconditioned with 50 mmol L−1 phosphate-buffer solution (PBS) at pH 8.1. Sample solution (RB, SR: 0.5 mmol L−1, F-BSA: 4 mg mL−1) and two buffer solutions for diffusion-based separation and sample injection were introduced by use of syringe pumps with appropriate flow rates (see figure captions). Sample injection was achieved by on–off switching of the syringe pump delivering buffer 2, shown in Fig. 1. Immediately after injection, high voltage was applied manually by use of a standard high-voltage power supply (Matsusada Precision, Shiga, Japan).
Capturing fluorescence images and laser-induced fluorescence measurement
Fluorescence images of the microchannel were obtained by using an optical/fluorescence inverted microscope (Eclipse TS100-F, Nikon, Tokyo, Japan). Photographs were captured using a 3CCD color camera (HV-D28S, Hitachi Kokusai Electric, Tokyo, Japan) installed at the front port of the microscope. Fluorescent images were collected using a mercury lamp as a light source and a filter block (G-2A and FITC, Nikon, Tokyo, Japan).
CE with laser-induced fluorescence (LIF) detection was performed on a home-built system based on an inverted fluorescence microscope (IX70, Olympus, Tokyo, Japan). Light at 488 nm from an argon ion laser (Newport Spectra Physics Laser Division, Mountain View, CA, USA) was introduced into the microscope. The laser beam was filtered through a 460–490 nm band-pass filter, reflected by a 510 nm dichroic mirror, then focused on the detection point by means of a 20× objective lens. Fluorescence was collected by use of the same objective lens, filtered through a 515 nm high-pass filter, and finally detected by use of a CCD camera (Model PMA-11, Hamamatsu Photonics, Shizuoka, Japan). To obtain electropherograms fluorescence at 600 nm was used throughout.
Results and discussion
Optimization of diffusion-based deproteinization
Diffusion-based separation of chemical species by use of multilayer flow was first reported by Brody et al. [23]. They separated small molecules from a particle-containing sample, or proteins from a biological cell-containing sample [23, 24]. Here we used this technique to separate small molecules from a mixed protein sample. When the sample solution containing small molecules and proteins made contact with the buffer-solution flow, the small molecules, which have larger diffusion coefficients than the proteins, diffused into the buffer solution. In this study, RB and SR were used as models for small molecules and F-BSA as that for a large molecule.
Figure 3 shows the fluorescence images at the confluence point of two flows and the injection point. Fluorescence images for F-BSA and rhodamine molecules were captured using different optical filters. In this case, buffer 2 was not flowing. As can be seen, when the total flow rate of sample and buffer was very high, neither F-BSA nor rhodamine molecules could reach the separation capillary (Fig. 3a). In contrast, selection of an appropriate total flow rate resulted in selective diffusion of small rhodamine molecules and negligibly small protein diffusion (Fig. 3b). Figure 3b also shows the fluorescence intensity profiles obtained by analysis of the fluorescence images for the rhodamine derivatives. According to this, the fluorescence intensity of the rhodamine-based molecules at the injection point was approximately 40% of the initial fluorescence intensity at the confluence point (see fluorescence intensity at position y at the injection point shown in the fluorescence intensity profile). Because complete diffusion gives 50% signal intensity for rhodamine-based molecules, these results suggest that diffusion of the rhodamine-based molecules was almost complete. In our experiments, surface adsorption of the solutes by the channel surface occurred, because of the hydrophobicity of the PDMS surface. Because these samples were continuously flowing for deproteinization, however, surface adsorption did not seriously affect the deproteinization procedure when sample injection was performed after steady flow was achieved.
Fig. 3Fluorescence images obtained at the confluence and injection points at different flow rates. (a) Total flow rate 10 μL min−1 (sample 5 μL min−1, buffer 1 5 μL min−1). (b) Total flow rate 1 μL min−1 (sample 0.5 μL min−1, buffer 1 0.5 μL min−1). In (b), fluorescence intensity profiles obtained by analysis of the fluorescence images obtained for rhodamine derivatives (SR, RB) are also shown. F-BSA and rhodamine-based molecules were detected by use of different optical filters
In this study the molecular weights of RB and SR are 443 and 581, respectively, and that for F-BSA is approximately 73,000 [25]. According to the literature, the diffusion coefficient for a small molecule with a molecular weight of 342 (sucrose) is 5.2×10−6 (cm2 s−1) and that for a large molecule with a molecular weight of 69,000 (HSA) is 6.1×10−7 (cm2 s−1) [26, 27]. Because these molecular weights are very close to those of the molecules used in this study, an approximately tenfold difference between the diffusion coefficients of the small and large molecules enabled successful separation of small molecules from mixed protein sample solution. Although the difference between diffusion coefficients is a primary principle of diffusion-based separation, it should be noted that the optimum conditions may change, depending on the concentration ratio of the protein and small molecules. Therefore, careful optimization experiments may be required when the concentrations of the small molecules are very low.
Sample injection and electrophoretic separation
Sample injection was performed manually by on–off switching of the syringe pump delivering buffer 2. In the diffusion-based separation and the CE separation mode, three-layer flow composed of sample solution, collection buffer (buffer 1), and buffer 2 occurs in the 300 μm square-PDMS channel along the buffer 2 flow channel, as illustrated in Fig. 1. Because buffer 2 prevents introduction of sample solution into the separation capillary, on-off switching of the syringe pump (buffer 2) enables sample injection similar to the “gated injection” reported by Jacobson et al. [28]. Usually, stopping the syringe pump does not immediately stop flow of buffer 2. In this work, therefore, it took several seconds to start injection. This causes diffusion of molecules at the front and rear ends of the sample plug. When sequential sample injections with injection times of 15 s were performed, however, fairly good reproducibility of 6.5% RSD (peak height; n=5) was obtained. Although this method of injection needs to be improved, good reproducibility led us to use this simple method for further investigation.
Figure 4 shows detection signals obtained at 10 cm downstream of the injection point with and without voltage application. When the sample plug is introduced without voltage application, ca. 160 s, corresponding to a linear flow rate of 0.63 mm s−1, was required for detection. The approximate linear flow rate of the pressure-driven flow inside the separation capillary can be estimated from the total flow rate of sample and buffer flow (in total 2 μL min−1), and the ratio of the cross sectional areas of the PDMS channel (300 μm square) and the separation capillary (50 μm square). Under our experimental conditions the calculated linear flow rate was ca. 0.36 mm s−1. Although the order of the calculated value was the same as the experimental value, the difference may be because of the slight difference between the back pressures of the two waste reservoirs. When the high voltage of 5 kV (417 V cm−1) was applied just after injection, the time required for detection was reduced to ca. 70 s and slight separation was observed. Because the net charge of SR was −1 and that of RB was 0, migration time of SR was slightly longer than that of RB. These results indicated that induction of electroosmotic flow and separation by electrophoresis, by voltage application, were confirmed.
Fig. 4Detection of rhodamine-based molecules (RB and SR) downstream of the separation capillary with and without application of high voltage. Flow rates: sample, 1 μL min−1; buffer 2, 1 μL min−1. Injection time: 20 s
Deproteinization and electrophoretic separation
On the basis of these experiments, deproteinization and electrophoretic separation were achieved. Figure 5 shows the electropherograms obtained at the downstream of the separation capillary with and without diffusion-based deproteinization. When the sample solution containing RB, SR, and F-BSA was introduced into the separation capillary without deproteinization, a broad signal arising from F-BSA was observed after the appearance of the RB and SR peaks (Fig. 5a). This broad signal may be because of the protein adsorption frequently observed in CE, or the multiple labeling of the F-BSA used in this work [29]. Commercially available F-BSA contains 7–12 labeling molecules (average) per BSA molecule. Therefore, the electrophoretic mobilities of BSA species with different numbers of labels may cause the broad signals shown in Fig. 5a. In contrast, the electropherogram obtained after deproteinization by use of the multilayer flow had a completely flat baseline after the appearance of two peaks of the rhodamine molecules (Fig. 5b). Thus our system enabled successful diffusion-based deproteinization and CE separation. Although interference from the protein was successfully removed, resolution of RB and SR is still not good in Fig. 5b (resolution: R=1.4). When the longer, 48 cm, separation capillary (effective length: 33 cm) was connected to the CAs-CHIP instead of 12 cm capillary, however, baseline separation of these species after deproteinization was successfully achieved (resolution: R=5.7). In addition, relative standard deviations of peak height and migration time for five sequential measurements were 11.4 and 11.5% for RB and SR, and 1.5 and 1.4% for RB and SR, respectively, indicating that reliable analysis with deproteinization pretreatment was successfully achieved by use of a longer separation capillary.
Fig. 5Electropherograms obtained downstream of the separation capillary with and without diffusion-based deproteinization. Flow rates: sample, 0.5 μL min−1, buffer 1 0.5 μL min−1, buffer 2 1 μL min−1. Injection time 15 s. Applied voltage 5 kV (ca. 417 V cm−1)
Conclusions
We have demonstrated deproteinization and CE separation on a single device using CAs-CHIP technology. Diffusion-based deproteinization in a CAs-CHIP was successfully achieved by choosing an appropriate flow rate, and subsequent CE separation of small molecules was achieved by use of a separation capillary connected directly to the CAs-CHIP.
Because diffusion-based separation is, in general, based on dilution of the sample solution, preconcentration before CE separation will be required in the next step. Because the CAs-CHIP is prepared by simply embedding square capillaries, however, further integration of the preconcentration process using chemically-functionalized capillaries, or the on-line preconcentration techniques reported to date, can be also used. These applications are currently under investigation.

capillary-assembled microchip
deproteinization
capillary electrophoresis
square capillary
polydimethylsiloxane


AVATAR: A database for genome-wide alternative splicing event detection using large scale ESTs and mRNAs

In the past years, identification of alternative splicing (AS) variants has been gaining momentum. We developed AVATAR, a database for documenting AS using 5,469,433 human EST sequences and 26,159 human mRNA sequences. AVATAR contains 12000 alternative splicing sites identified by mapping ESTs and mRNAs with the whole human genome sequence. AVATAR also contains AS information for 6 eukaryotes. We mapped EST alignment information into a graph model where exons and introns are represented with vertices and edges, respectively. AVATAR can be queried using, (1) gene names, (2) number of identified AS events in a gene, (3) minimal number of ESTs supporting a splicing site, etc. as search parameters. The system provides visualized AS information for queried genes.
Background
Alternative splicing (AS) is an important mechanism for functional diversity in eukaryotic cells. AS allow processing
of one pre-mRNA into different transcripts in a cell type. This results in protein diversity with each protein having
distinct function. [1–2
–3] To address this problem we used EST (short, single pass cDNA
sequences generated from randomly selected library clones produced in a high throughput manner from different tissues,
individuals and conditions) and mRNA sequences to detect AS variants. The detected variants (using 5,469,433 EST and
26,159 mRNA sequences) were stored in a database called AVATAR.
Although, AS databases are available in the public domain, not many contain AS information for multiple eukaryotes
(a comparison summarized in AVATAR web site). Therefore, it is important to document AS information for multiple
eukaryotes. Hence, we developed AVATAR containing AS information for six eukaryotes. Here, we describe AVATAR development,
its content and utility.
Methodology
Dataset used
The dbEST database (Jan 16, 2004) at NCBI contains nearly 5.4 million human EST sequences and this dataset is used
in the current analysis. [4] The human genome sequences (CONTIG build 3.4)
in Genbank format is obtained from NCBI. [5] Gene information and
mRNA sequence were downloaded from the NCBI RefSeq project.
Identification of AS
The identification of AS in AVATAR is performed in three steps (described below) as illustrated in Figure 1.
Step 1:
Alignment of EST and mRNA with the genome sequence
EST sequences were aligned to the whole genome sequence using Mugup. [6
] Mugup is a sequence  alignment program developed in Windows platform. This procedure identified splice sites in
the ESTs (Figure 1 panel A and B). The matched regions and gaps correspond to exons and introns, respectively. EST and mRNA
alignments with scores greater than 94% were used for further analysis.
Step 2:
Clustering EST and mRNA
EST and mRNA were clustered according to their location in the genome (Figure 1 panel C). EST and mRNA with overlapping
regions were then assembled together.
Step 3:
Detection of AS sites
The mapping of EST alignment with genome sequence to intron positions helps to identify skipped exons and
included exons.
Searching AVATAR
AVATAR can be queried using keywords. The keywords include accession number, gene name, gene isoform, gene location,
cytogenetic locations, chromosome number and number of AS events. The database search produces AS visuals for queried gene.
Utility to the Biological Community
AVATAR is a collection of AS information for 6 eukaryotic organisms. The database can be queried simultaneously for 6
organisms. It can also be searched using gene names and desired number of AS events. EST sequences are error prone resulting
in the detection of aberrant transcripts. Frequency of EST alignment at a specific site provides improved detection in AVATAR.
Caveats
AS information on paralogous genes in eukaryotic genomes are not included in AVATAR due to the difficulty in identifying
their corresponding chromosomal locations using EST sequences.
Future developments
New EST sequences are generated in laboratories every day. Hence, it is a time consuming to keep AS databases updated due
to the growth of genome and mRNA sequences. Hence, we are in the process of developing a computer agent which can update AVATAR
automatically. We also plan to include tumor specific AS data.

database
alternative splicing
est
mrna
human
eukaryotes
protein diversity
sequence alignment


High failure rate of the interspinous distraction device (X-Stop) for the treatment of lumbar spinal stenosis caused by degenerative spondylolisthesis

The X-Stop interspinous distraction device has shown to be an attractive alternative to conventional surgical procedures in the treatment of symptomatic degenerative lumbar spinal stenosis. However, the effectiveness of the X-Stop in symptomatic degenerative lumbar spinal stenosis caused by degenerative spondylolisthesis is not known. A cohort of 12 consecutive patients with symptomatic lumbar spinal stenosis caused by degenerative spondylolisthesis were treated with the X-Stop interspinous distraction device. All patients had low back pain, neurogenic claudication and radiculopathy. Pre-operative radiographs revealed an average slip of 19.6%. MRI of the lumbosacral spine showed a severe stenosis. In ten patients, the X-Stop was placed at the L4–5 level, whereas two patients were treated at both, L3–4 and L4–5 level. The mean follow-up was 30.3 months. In eight patients a complete relief of symptoms was observed post-operatively, whereas the remaining 4 patients experienced no relief of symptoms. Recurrence of pain, neurogenic claudication, and worsening of neurological symptoms was observed in three patients within 24 months. Post-operative radiographs and MRI did not show any changes in the percentage of slip or spinal dimensions. Finally, secondary surgical treatment by decompression with posterolateral fusion was performed in seven patients (58%) within 24 months. In conclusion, the X-Stop interspinous distraction device showed an extremely high failure rate, defined as surgical re-intervention, after short term follow-up in patients with spinal stenosis caused by degenerative spondylolisthesis. We do not recommend the X-Stop for the treatment of spinal stenosis complicating degenerative spondylolisthesis.
Introduction
Lumbar spinal stenosis complicating degenerative spondylolisthesis is a common cause for low back pain, neurogenic claudication, and radiculopathy in the elderly population. The majority of the patients will respond well to non-operative treatment modalities. However, in patients that fail to respond to conservative treatment, surgical decompression with or without a posterolateral fusion and instrumentation, may be considered [3, 12]. Unfortunately, these procedures have variable long-term outcomes and are frequently followed by complications, especially in the elderly patients with high co-morbility [2, 10]. Therefore, alternative therapies are being developed, of which the interspinous distraction device is rapidly gaining popularity [4, 9]. Of such, the X-Stop (X-Stop, St. Francis Medical Technologies, Inc®, Alameda, CA) has been introduced as a minimal invasive surgical procedure to treat symptomatic degenerative lumbar spinal stenosis [4, 9]. Initial results of the treatment of degenerative lumbar spinal stenosis with the X-Stop are promising [8, 13, 14]. Recently, encouraging results have been reported for the treatment of patients with symptomatic lumbar spinal stenosis caused by degenerative spondylolisthesis [1]. However, we observed an alarmingly high failure rate, defined as surgical re-intervention, in a cohort of patients treated with the X-Stop for symptomatic lumbar spinal stenosis caused by degenerative spondylolisthesis. This prompted us to perform a retrospective chart review, and analysis of the radiographs.
Patients and methods
We retrospectively reviewed 12 consecutive patients with symptomatic lumbar spinal stenosis caused by degenerative spondylolisthesis treated with the X-Stop interspinous distraction device. The patients were treated between January 2003 and May 2005. There were 9 female and 3 male patients with a mean age at surgery of 67.5 years (50–83). All patients complained of progressive low back pain throughout the day with neurogenic claudication, radiculopathy and a diminished walking distance. In all patients, neurological examination was judged normal or nonspecific. Anteroposterior, lateral and flexion/extension plain radiographs, and magnetic resonance imaging (MRI) were performed in all cases. The percentage of degenerative slip was measured on the lateral radiograph and measured according to the method described by Anderson et al. [1]. The anteroposterior dural sac diameter in the axial and sagittal plane T2 sequence was measured on the MRI. A standardized walking and cycling test [6] was performed at the department of physical therapy. A limited walking distance less than 1 km (0.62 miles) independent of the time needed, was considered positive. After walking the patient had to sit and the pre-walking pain must be reduced in less than 5 min. Cycling should be unlimited without complaints. Initial treatment consisted on patient education, medications to control pain, and exercise and physical treatments to regain or maintain activities of daily living. Surgical treatment with the X-Stop was considered in patients not improving with conservative care for more than 6 months.
All operations were performed under general anesthesia. The patients were placed in prone position on a Wilson spinal surgery frame (Orthopaedic Systems, Inc., Union City, CA) with the lumbar spine in maximum flexion. Prophylactic antibiotics, cefazolin (cefalosporin, Kefzol®) 1,000 mg IV, were administered at the induction of anesthesia, and as a second and third dose 8 and 16 h post-operatively, respectively. After radiographic identification of the surgical level, a mid-sagittal incision of approximately 4 cm is made over the spinous processes. The musculature was elevated to the level of laminae and facets. The supraspinal ligament is kept intact. To pierce the interspinous ligament, a curved dilator is inserted in the anterior margin of the interspinous space. Subsequently, a sizing distractor is inserted to determine the appropriate implant size. The X-Stop is inserted into the interspinous space as close to the posterior aspect of the lamina as possible. An adjustable wing is attached to the implant and secured along the midline. Patients were mobilized immediately once they had recovered from the anesthetic effects. They were discharged from hospital within 48 h.
Clinical follow-up took place at 6 and 12 weeks and at 12 and 24 months. All patients underwent a clinical and radiographic examination of the lumbar spine in standing position at each follow-up visit. The mean follow-up was 30.3 months (13–41). In patients with persistent or recurrence low back pain with neurogenic claudication and radiculopathy, a second MRI was made. The endpoint was secondary surgical intervention of the lumbar spine. Statistical analysis, comparing the pre- en post-operative MRI dimensions, was performed using Students’ t-test.
Results
The pre-operative percentage of degenerative spondylolisthesis was less than 30% in all patients, with an average slip of 19.6% ± 6.20 (9.6–29.7). In 9 out of the 12 patients there was a slip of less than 25% (grade 1) degenerative spondylolisthesis. Bending radiographs revealed mobility at the level of the spondylolisthesis in all patients. MRI showed nerve root compression and impingement of the thecal sac. The mean anteroposterior axial cross-sectional diameter was 7.33 mm (5.71–11.19) and the mean anteroposterior sagittal cross-sectional diameter was 7.32 mm (5.40–8.49).
The operations were performed at L4–5 in ten of the patients and at both L3–4 and L4–5 in two patients. A 14-mm diameter X-Stop was implanted in nine levels. In the remaining levels, a 12-mm implant was used three times, and 16 and 10-mm implants both once. No peri-operative complications were observed. Post-operative plain radiographs showed a correct position of the implants in all patients. No fractures of the spinous processes were observed. The post-operative percentage of spondylolisthesis, measured on plain radiographs post-operatively and at final follow-up, remained unchanged in all patients.
Direct post-operatively, 8 out of 12 patient reported a significant improvement of pain, neurogenic claudication, and radiculopathy. However, four patients did not experience any relief of symptoms following surgery and no improvement at follow-up. At 12 weeks follow up, two patients, that initially had experienced a relief of symptoms suffered from a recurrence of pain, neurogenic claudication, and radiculopathy. In addition, a third patient experienced a recurrence of symptoms at 24 months follow-up.
All patients with persistent or recurrent symptoms had a post-operative MRI. No statistically significant (P > 0.05) difference of spinal stenosis was seen at the effected levels in comparison to the pre-operative values. The mean post-operative anteroposterior axial cross sectional diameter was 6.80 mm (5.24–7.65) and the mean sagittal cross sectional diameter was 6.91 mm (5.12–7.70) (Fig. 1, 2, 3). The pre-operative axial and sagittal cross sectional diameter in these seven patients (7 levels) was not significantly different (P > 0.05) from that of the five patients (7 levels) without persistent or recurrent symptoms. Finally, the seven patients with persistent or recurrent symptoms underwent surgical re-intervention. The mean degenerative spondylolisthesis of these seven patients was 17.8% ± 6.9. Six of these patients had a pre-operative degenerative spondylolisthesis of less than 25%. One patient had a 27.6% degenerative spondylolisthesis. The X-Stop was removed and a decompression and posterolateral fusion with instrumentation was performed.
Fig. 1a Pre-operative lateral plain radiograph. b Post-operative lateral plain radiograph. X-Stop positioned at the level L4–5Fig. 2Pre-operative T2-weighted a transversal and b sagittal MR Image showing lumbar spinal stenosis due to discopathy, facet arthritis, ligamentum flavum hypertrophy and anterolisthesisFig. 3Post-operative T2-weighted a transversal and b sagittal MR Image. No change in canal cross-sectional area and mid-sagittal diameter visible after insertion of the X-Stop at level L4–5
Discussion
The X-Stop interspinous distraction device has shown to be an attractive alternative to conventional surgical procedures in the treatment of symptomatic degenerative lumbar spinal stenosis [4, 9]. It may be questioned, however, if the X-stop will be effective in patients with lumbar spinal stenosis caused by degenerative spondylolisthesis also. To our best knowledge, there is only one study that investigated the clinical effects of the X-Stop in patients with lumbar spinal stenosis caused by degenerative spondylolisthesis [1]. In this study, 42 patients were treated with the X-Stop and compared to 33 patients with non-operative treatment. The indication for treatment was a percentage of slip of less than 25%. An overall clinical success rate of 63.4% was reported in the X-Stop treated patients compared to 12.9% in the non-operative treated patients after 2 year follow-up. Secondary surgery was required in 5 (11.9%) of the patients in the X-Stop group compared to 4 (12.1%) in the control group.
Unfortunately, we experienced an extremely high failure rate, defined as surgical re-intervention, in a cohort of patients with lumbar spinal stenosis caused by degenerative spondylolisthesis treated with the X-Stop interspinous distraction device. In our cohort, the average percentage of slip was less than 25%, though in 3 patients the percentage of slip was between 25 and 30%. Surgical re-intervention was required in 7 (58%) patients within 24 months. Of these, only 1 patient had a slip of more than 25% (27.6%). There was no relation between the severity of the slip and the failures in our cohort. Our indication for re-intervention included recurrence or persistent and unremitting low back pain and persistent or progressive neurogenic claudication with radiculopathy. Both clinical and radiological findings were considered together for diagnosing failure of treatment. Unfortunately, we did not enclose pre- and post-operative outcome measurements. However, since the surgical goal of the X-Stop include pain reduction, improvement of neurological symptoms, and improvement of quality of live, re-intervention was considered as the endpoint for failure.
In diagnosing spinal stenosis, thecal sac impingement and nerve root compression are seen on MRI. We observed no improvement of the axial and sagittal diameter of the central canal on the MRI after insertion of the X-Stop. In addition, no relation was found between the severity of the pre-operative spinal stenosis measured on MRI and an eventual secondary surgical intervention. Recently, in a study using positional MRI pre- and post-operatively following insertion of the X-Stop, improvement of the cross sectional area of the dural sack has been observed in 12 patients with symptomatic spinal stenosis [11]. This study, however, did not include patients with spinal stenosis caused by degenerative spondylolisthesis. Unfortunately, we do not have the opportunity to use the positional MRI. It may be hypothesized that the spinal stenosis will be more severe in a standing positional MRI, as a result of the instability in degenerative spondylolisthesis. A limitation of the present study is the lack of objective standards of measurement spinal stenosis on MRI. Nevertheless, all patients in our study showed pre-operative a severe thecal sack impingement at the level of degenerative spondylolisthesis. In addition, the spondylolisthesis, as measured on the lateral standing radiographs, did not show progression or improvement after surgery.
From a biomechanical point of view, it may be questioned if the X-Stop interspinous distraction device provides any stabilizing effect on the affected motion segment and will increase the spinal canal in degenerative spondylolisthesis. It has been shown that the facet joints in patients with degenerative spondylolisthesis demonstrate an increased sagittal orientation [5, 7]. When the facet joints are orientated in a more sagittal plain, the resistance to shear forces is decreased. Obviously, the more sagittal orientation of the L4–L5 segment combined with an interspinous distraction device may result in a progressive forward slip of the superior vertebra, and a progressive narrowing of the spinal canal and lateral recesses. Thus, the presence of a degenerative spondylolisthesis in patients with lumbar spinal stenosis may be considered as a contra indication for the X-Stop.
In conclusion, the X-Stop interspinous distraction device showed an extremely high failure rate, defined as surgical re-intervention, after short term follow-up in patients with spinal stenosis caused by degenerative spondylolisthesis. We do not recommend the X-Stop for the treatment of lumbar spinal stenosis with degenerative spondylolisthesis, and we consider a degenerative spondylolisthesis a contra-indication for the X-Stop interspinous distraction device.
Competing interests
No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

x-stop
lumbar spinal stenosis
degenerative spondylolisthesis