Functional Spacers Research Articles

Evaluating Modern Spacer Options and Outcomes in Revision Hip Arthroplasty

BackgroundA 2-stage revision continues to be the standard treatment for periprosthetic joint infection (PJI) in hip arthroplasty. The use of “functional” spacers may allow patients to return to daily living while optimizing their health for revision surgery. We aimed to evaluate the clinical outcomes of different spacer types regarding infection eradication, mechanical complications, and functional outcomes. MethodsPatients who have complete Musculoskeletal Infection Society criteria for diagnosis of PJI that underwent one-stage or 2-stage revision were queried in an institutional surgical database between 2002 and 2022. Out of 286 patients, 210 met our inclusion criteria and were retrospectively reviewed for demographics, laboratory values, functional and patient-reported outcomes, and subsequent revisions. The study population had 54.3% women, a mean age of 61 years old, and a mean follow-up of 3.7 ± 3.2 years. There was no difference between age, body mass index, or Charlson Comorbidity Index scores between each cohort. Spacers were categorized as nonfunctional static, nonfunctional articulating, or functional articulating. Functional spacers were defined as those that allowed full weight bearing with no restrictions. Delphi criteria were used to define revision success, and failure was defined as a recurrent or persistent infection following definitive surgery. ResultsThere was a significantly lower reoperation rate after a definitive implant in the functional articulating cohort (P = .003), with a trending higher infection eradication rate and a lower rate of spacer failure compared to the nonfunctional spacer cohort. At 5 years, functional articulating spacers had a 94.1% survivorship rate, nonfunctional articulating spacers had an 81.2% survival rate, and nonfunctional static spacers had a 71.4% survival rate. In the functional articulating spacer cohort, 14.6% had yet to get reimplanted, with an average follow-up time of 1.4 years. ConclusionsWithin this large cohort of similar demographics, functional articulating spacers may result in better clinical outcomes and infection eradication during 2-stage revision arthroplasty for PJI.

Modern Description of Antibiotic Spacer Options for PJI in Revision Total Knee Arthroplasty: Clinical Outcomes and Infection Eradication Rates

BackgroundAs the burden of periprosthetic joint infections (PJIs) increases, there is growing interest in understanding the efficacy and morbidity reduction of 2-stage revision and various antibiotic spacer options. This study aimed to expand the description and evaluation of spacers from solely their articulation status to include their ability to support full (functional) or partial weight-bearing (nonfunctional). MethodsBetween 2002 and 2021, 391 patients who had Musculoskeletal Infection Society criteria for PJI with 1-stage or 2-stage revision were included. Demographics, functional outcomes, and subsequent revision data were collected. The study population had a mean follow-up of 2.9 years (range, 0.05-13.0) with an average age of 67 years (range, 34.7-93.4). Spacer failure was defined by surgical intervention following definitive surgery, and infection eradication was defined by the Delphi criteria. Spacers were classified as nonfunctional static, nonfunctional dynamic, functional static, or functional dynamic. Two tailed t-tests were performed. ResultsThere were no significant differences in infection eradication or mechanical outcomes across spacer types; notably, 97.3% of functional dynamic spacers achieved infection eradication. Functional spacers had a longer time to the second stage procedure and a greater number of patients who had not been reimplanted. There was no difference in reoperation rates in nonfunctional versus functional spacers. ConclusionWithin this cohort, infection eradication and spacer exchange rates were noninferior among spacers. Functional spacers may allow for earlier return to daily living given the weight-bearing capability when compared to nonfunctional, without sacrificing clinical outcome.

PAM binding ensures orientational integration during Cas4-Cas1-Cas2-mediated CRISPR adaptation

Adaptation in CRISPR-Cas systems immunizes bacteria and archaea against mobile genetic elements. In many DNA-targeting systems, the Cas4-Cas1-Cas2 complex is required for selection and processing of DNA segments containing PAM sequences prior to integration of these "prespacer" substrates as spacers in the CRISPR array. We determined cryo-EM structures of the Cas4-Cas1-Cas2 adaptation complex from the type I-C system that encodes standalone Cas1 and Cas4 proteins. The structures reveal how Cas4 specifically reads out bases within the PAM sequence and how interactions with both Cas1 and Cas2 activate Cas4 endonuclease activity. The Cas4-PAM interaction ensures tight binding between the adaptation complex and the prespacer, significantly enhancing integration of the non-PAM end into the CRISPR array and ensuring correct spacer orientation. Corroborated with our biochemical results, Cas4-Cas1-Cas2 structures with substrates representing various stages of CRISPR adaptation reveal a temporally resolved mechanism for maturation and integration of functional spacers into the CRISPR array.

A Campylobacter integrative conjugative element with a CRISPR-Cas9 system targeting competing plasmids: a history of plasmid warfare?

Microbial genomes are highly adaptable, with mobile genetic elements such as integrative conjugative elements (ICE) mediating the dissemination of new genetic information throughout bacterial populations. This is countered by defence mechanism such as CRISPR-Cas systems, which limit invading mobile elements by targetting specific sequences on these elements. Here we have studied the distribution the pVir, pTet and PCC42 plasmids and a new 70-129 kb ICE (CampyICE1) in the foodborne microbial pathogens Campylobacter jejuni and Campylobacter coli. CampyICE1 contains a degenerated Type II-C CRISPR system consisting of a sole Cas9 protein, which is distinct from the previously described Cas9 proteins from C. jejuni andC. coli. CampyICE1 is highly conserved in structure and gene order, containing blocks of genes predicted to be involved in recombination, regulation and conjugation. CampyICE1 was detected in 134/5,829 (2.3%) C. jejunigenomes and 92/1,347 (6.8%) C. coligenomes. Similar ICE were detected in a number ofnon-jejuni/coli Campylobacter species, which lacked a CRISPR-Cas system. Finally, CampyICE1 contained 3 separate short CRISPR spacer arrays, and a total of 124 unique spacers were identified, of which 67 are predicted to target the Campylobacterplasmids pVir, pTet, and pCC42, and 12 predicted to target other Campylobacter plasmids (63.7%). The presence of a functional CampyICE1 Cas9 protein and matching anti-plasmid spacers was associated with the absence of these plasmids (186/214 genomes), implicating that the CampyICE1-encoded CRISPR-Cas has contributed the exclusion of competing plasmids. Hence the CampyICE1 CRISPR-Cas system may be a part of ongoing plasmid warfare in Campylobacter spp.

Adaptive antimicrobial resistance, a description of microbial variants, and their relevance to periprosthetic joint infection.

Periprosthetic joint infection (PJI) is a difficult complication requiring a comprehensive eradication protocol. Cure rates have essentially stalled in the last two decades, using methods of antimicrobial cement joint spacers and parenteral antimicrobial agents. Functional spacers with higher-dose antimicrobial-loaded cement and antimicrobial-loaded calcium sulphate beads have emphasized local antimicrobial delivery on the premise that high-dose local antimicrobial delivery will enhance eradication. However, with increasing antimicrobial pressures, microbiota have responded with adaptive mechanisms beyond traditional antimicrobial resistance genes. In this review we describe adaptive resistance mechanisms that are relevant to the treatment of PJI. Some mechanisms are well known, but others are new. The objective of this review is to inform clinicians of the known adaptive resistance mechanisms of microbes relevant to PJI. We also discuss the implications of these adaptive mechanisms in the future treatment of PJI. Cite this article: Bone Joint J2022;104-B(5):575-580.

Understanding the role of Cas4 during adaptation in CRISPR‐Cas immune systems

CRISPR-Cas systems (Clustered regularly interspaced short palindromic repeats and associated proteins) are adaptive immune systems that protect bacteria and archaea against invading phages. Spacer sequences derived from invader genome are arranged between repeat sequences in the CRISPR array. Upgrading the CRISPR array with new spacers is a crucial step to ensure bacterial systems are at pace with rapidly evolving phages. This is carried out by Cas1 and Cas2 proteins, universally present in most CRISPR systems. Many systems require proteins in addition to Cas1 and Cas2 to maintain the efficiency and fidelity of adaptation. Cas4 is one such protein and is part of the core family of Cas proteins in over half of known CRISPR systems. Cas4 carries a RecB like nuclease domain and initial studies on Cas4 characterized its function as a 5’ to 3’ exonuclease. Recent in-vivo and in-vitro studies have shown that Cas4 plays a role in selection, processing, and integration of functional spacers. Our lab has shown that Cas4 performs endonucleolytic processing of 3’ overhangs of prespacers in the presence of Cas1 and Cas2. Using negative stain EM, we have shown that Cas4 forms a complex with Cas1 and Cas2 in the presence of a potential spacer substrate. However, the exact mechanistic steps in Cas4-Cas1-Cas2 mediated integration remain unknown. Moreover, some systems encode Cas4-1 fusion proteins that provide an increased efficiency of spacer acquisition observed in-vivo. These fusions remain to be characterized biochemically. With this work, we aim to determine the sequence of events in integration in systems that have Cas4 and understand the structure function relationship of Cas4 in fusion with Cas1 and complex with Cas1-Cas2.

Selective loading and processing of prespacers for precise CRISPR adaptation

CRISPR-Cas immunity protects prokaryotes against invading genetic elements1. It uses the highly conserved Cas1-Cas2 complex to establish inheritable memory (spacers)2-5. How Cas1-Cas2 acquires spacers from foreign DNA fragments (prespacers) and integrates them into the CRISPR locus in the correct orientation is unclear6,7. Here, using the high spatiotemporal resolution of single-molecule fluorescence, we show that Cas1-Cas2 selects precursors of prespacers from DNA in various forms-including single-stranded DNA and partial duplexes-in a manner that depends on both the length of the DNA strand and the presence of a protospacer adjacent motif (PAM) sequence. We also identify DnaQ exonucleases as enzymes that process the Cas1-Cas2-loaded prespacer precursors into mature prespacers of a suitable size for integration. Cas1-Cas2 protects the PAM sequence from maturation, which results in the production of asymmetrically trimmed prespacers and the subsequent integration of spacers in the correct orientation. Our results demonstrate the kinetic coordination of prespacer precursor selection and PAM trimming, providing insight into the mechanisms that underlie the integration of functional spacers in the CRISPR loci.

Non-adiabatic annular flow experiments in a subchannel geometry with spacer grid

Boiling annular flow in a subchannel geometry with functional spacers is studied using X-Ray tomography in the Dryout Tomography Experiment (DoToX) facility at ETH Zürich. The liquid film distribution on the walls and the liquid holdup within the gas core of the annular flow in the subchannel, both under the influence of the spacer vanes, are extracted from tomographic images. The subchannel cross section consists of a main subchannel in the center surrounded by four heating flow sections representing fuel rods and connected to four partial subchannels with simplified geometry. This effectively removes unnatural no-slip boundary conditions at the narrow gap of the main subchannel.Chloroform is used as a working fluid because of its low enthalpy of evaporation and a low boiling point around 60 °C close to atmospheric pressure. A liquid heating system with water at slightly above 100 °C provides the heating power for the boiling process. This ensures continuous operation of the facility even in case of a dryout scenario, avoiding structural damages by imposing a maximum system temperature. The high thermal capacity of the water allows for an almost isothermal boundary condition. Although not identical to real BWR settings, this experiment is well suited to generate data for model validation.Originally designed for tomographic imaging with both cold neutrons and X-rays, this paper focuses on results obtained by X-rays. Since the X-ray contrast between the working fluid chloroform and the structural material of the channel, aluminum, is very small, differential imaging with adaptive centering of reconstructions with and without the liquid film were required. The measurement campaign consisted of three measurements with constant chloroform mass flows at 5.00, 4.05 and 3.25 kg/min and constant heating liquid temperature of 100–104 °C, in addition to the empty channel reference measurement. The thermal power transferred to the working fluid under these conditions was about 10 kW of heating power. The resulting liquid film thicknesses (LFT) upstream of the spacer decreased from 91 to 49 µm. The effect of the spacer vanes is visualized and quantified over an axial section of 10 hydraulic diameters. Reference samples of the film thickness and the liquid holdup were taken upstream of the spacer. Dryout was nearly reached in characteristic regions of reduced film thickness due to turbulent shear stresses, while other regions show a significant local increase of the liquid film thickness. The deposition of about 30% of the liquid holdup from the gas core contributes to the increase. The results are consistent with previous adiabatic experiments with similar geometries.

The Cas4-Cas1-Cas2 complex mediates precise prespacer processing during CRISPR adaptation.

CRISPR adaptation immunizes bacteria and archaea against viruses. During adaptation, the Cas1-Cas2 complex integrates fragments of invader DNA as spacers in the CRISPR array. Recently, an additional protein Cas4 has been implicated in selection and processing of prespacer substrates for Cas1-Cas2, although this mechanism remains unclear. We show that Cas4 interacts directly with Cas1-Cas2 forming a Cas4-Cas1-Cas2 complex that captures and processes prespacers prior to integration. Structural analysis of the Cas4-Cas1-Cas2 complex reveals two copies of Cas4 that closely interact with the two integrase active sites of Cas1, suggesting a mechanism for substrate handoff following processing. We also find that the Cas4-Cas1-Cas2 complex processes single-stranded DNA provided in cis or in trans with a double-stranded DNA duplex. Cas4 cleaves precisely upstream of PAM sequences, ensuring the acquisition of functional spacers. Our results explain how Cas4 cleavage coordinates with Cas1-Cas2 integration and defines the exact cleavage sites and specificity of Cas4.

Cas4-Cas1 fusions drive efficient PAM selection and control CRISPR adaptation.

Microbes have the unique ability to acquire immunological memories from mobile genetic invaders to protect themselves from predation. To confer CRISPR resistance, new spacers need to be compatible with a targeting requirement in the invader's DNA called the protospacer adjacent motif (PAM). Many CRISPR systems encode Cas4 proteins to ensure new spacers are integrated that meet this targeting prerequisite. Here we report that a gene fusion between cas4 and cas1 from the Geobacter sulfurreducens I-U CRISPR–Cas system is capable of introducing functional spacers carrying interference proficient TTN PAM sequences at much higher frequencies than unfused Cas4 adaptation modules. Mutations of Cas4-domain catalytic residues resulted in dramatically decreased naïve and primed spacer acquisition, and a loss of PAM selectivity showing that the Cas4 domain controls Cas1 activity. We propose the fusion gene evolved to drive the acquisition of only PAM-compatible spacers to optimize CRISPR interference.

Cas4-Dependent Prespacer Processing Ensures High-Fidelity Programming of CRISPR Arrays

CRISPR-Cas immune systems integrate short segments of foreign DNA as spacers into the host CRISPR locus to provide molecular memory of infection. Cas4 proteins are widespread in CRISPR-Cas systems and are thought to participate in spacer acquisition, although their exact function remains unknown. Here we show that Bacillus halodurans type I-C Cas4 is required for efficient prespacer processing prior to Cas1-Cas2-mediated integration. Cas4 interacts tightly with the Cas1 integrase, forming a heterohexameric complex containing two Cas1 dimers and two Cas4 subunits. In the presence of Cas1 and Cas2, Cas4 processes double-stranded substrates with long 3' overhangs through site-specificendonucleolytic cleavage. Cas4 recognizes PAM sequences within the prespacer and prevents integration of unprocessed prespacers, ensuring that only functional spacers will be integrated into the CRISPR array. Our results reveal the critical role of Cas4 in maintaining fidelity during CRISPR adaptation, providing a structural and mechanistic model for prespacer processing and integration.

Cas4 Facilitates PAM-Compatible Spacer Selection during CRISPR Adaptation

SummaryCRISPR-Cas systems adapt their immunological memory against their invaders by integrating short DNA fragments into clustered regularly interspaced short palindromic repeat (CRISPR) loci. While Cas1 and Cas2 make up the core machinery of the CRISPR integration process, various class I and II CRISPR-Cas systems encode Cas4 proteins for which the role is unknown. Here, we introduced the CRISPR adaptation genes cas1, cas2, and cas4 from the type I-D CRISPR-Cas system of Synechocystis sp. 6803 into Escherichia coli and observed that cas4 is strictly required for the selection of targets with protospacer adjacent motifs (PAMs) conferring I-D CRISPR interference in the native host Synechocystis. We propose a model in which Cas4 assists the CRISPR adaptation complex Cas1-2 by providing DNA substrates tailored for the correct PAM. Introducing functional spacers that target DNA sequences with the correct PAM is key to successful CRISPR interference, providing a better chance of surviving infection by mobile genetic elements.

Development of neutron and X-ray imaging techniques for nuclear fuel bundle optimization

Abstract High-quality boiling crisis, i.e. dry out of the liquid film is an important operational safety issue for boiling water reactors. The phenomena involved in dry out are complicated and difficult to capture in full details in experiments. We follow a step-wise approach to the problem and utilize innovative, high-resolution measurement techniques. First extensive testing have been carried out on partial nuclear fuel bundle models containing typically a couple of neighboring subchannels. These first series featured adiabatic, air-water annular flows with and without the presence of prototypical functional spacers. Cold neutron imaging has been used to map the distribution of the liquid film thickness on the virtual fuel rods and on the spacer structures. As a next step a dedicated loop using a special working fluid has been designed enabling examining convective boiling two-phase flow in similar geometries as the adiabatic tests. Cold neutron and X-ray imaging can be used for the heated set-up to try to capture actual dry out phenomena. An overview of the result of the adiabatic test series and the first result with the heated experiments are given below.

Velocity Measurements and Turbulent Dispersion in Annular Liquid Film Flows in BWR Subchannels with Spacers

The thermal-hydraulic design characteristics of a boiling water reactor are strongly dependent on the physics of heat-removing mechanisms from nuclear rods. Performance is often limited by a lack of understanding of the heat and momentum transfer in thin liquid films close to dryout. This is particularly important with regard to power uprates of reactors. Flow control by functional spacers equipped with vanes adds complexity to the flow behavior. Instead of improving the heat removal, they can cause a local reduction of the film thickness under unfavorable conditions. This work presents a novel experimental technique to measure the velocity of the liquid phase and to study the turbulent mixing of a passive scalar in the film. The studies were performed in vertical annular flow in a double-subchannel geometry. The liquid film was labeled by either continuous or pulsed tracer injection. An electrically conductive tracer (salt solution) is injected into the film consisting of deionized water. The salt is traced using a liquid film sensor, a high-frequency nonintrusive conductivity-based technique. The experiments are conducted with and without a swirl-type spacer to quantify the effect of the spacers on the film flow for different gas and liquid flow rates. An ensemble averaging of the data obtained from individual injection pulses provides time-averaged liquid velocities in the film. The turbulent dispersion was characterized by analyzing the spread of the tracer in axial and lateral directions. Spacer vanes are found to induce a characteristic transverse movement of the liquid in the film. The effect of waves on mixing is analyzed and is found to enhance it.

Simulations and measurements of adiabatic annular flows in triangular, tight lattice nuclear fuel bundle model

High conversion light water reactors (HCLWR) having triangular, tight-lattice fuels bundles could enable improved fuel utilization compared to present day LWRs. However, the efficient cooling of a tight lattice bundle has to be still proven. Major concern is the avoidance of high-quality boiling crisis (film dry-out) by the use of efficient functional spacers. For this reason, we have carried out experiments on adiabatic, air-water annular two-phase flows in a tight-lattice, triangular fuel bundle model using generic spacers. A high-spatial-resolution, non-intrusive measurement technology, cold neutron tomography, has been utilized to resolve the distribution of the liquid film thickness on the virtual fuel pin surfaces. Unsteady CFD simulations have also been performed to replicate and compare with the experiments using the commercial code STAR-CCM+. Large eddies have been resolved on the grid level to capture the dominant unsteady flow features expected to drive the liquid film thickness distribution downstream of a spacer while the subgrid scales have been modeled using the Wall Adapting Local Eddy (WALE) subgrid model. A Volume of Fluid (VOF) method, which directly tracks the interface and does away with closure relationship models for interfacial exchange terms, has also been employed. The present paper shows first comparison of the measurement with the simulation results.

Cas9 specifies functional viral targets during CRISPR-Cas adaptation.

Clustered, regularly interspaced, short palindromic repeat (CRISPR) loci and their associated (Cas) proteins provide adaptive immunity against viral infection in prokaryotes. Upon infection, short phage sequences known as spacers integrate between CRISPR repeats and are transcribed into small RNA guides that identify the viral targets (protospacers) of the Cas9 nuclease. Streptococcus pyogenes Cas9 cleavage of the viral genome requires the presence of an NGG protospacer adjacent motif (PAM) sequence immediately downstream of the target. It is not known if and how viral sequences with the correct PAM are chosen as new spacers. Here we show that Cas9 specifies functional PAM sequences during spacer acquisition. The replacement of cas9 with alleles that lack the PAM recognition motif or recognize an NGGNG PAM eliminated or changed PAM specificity during spacer acquisition, respectively. Cas9 associates with other proteins of the acquisition machinery (Cas1, Cas2 and Csn2), presumably to provide PAM-specificity to this process. These results establish a new function for Cas9 in the genesis of the prokaryotic immunological memory.

Optimizing the performance of cold-neutron tomography for investigating annular flows and functional spacers in fuel rod bundles

In a previous study we used cold-neutron imaging to examine annular flows and the influence of functional spacers on such flows in a fuel bundle model. Investigating adiabatic, air–water annular flows in a scaled-up model of two neighboring subchannels of a BWR fuel assembly, we have shown that cold-neutron tomography is a promising tool to study such flows in nuclear fuel bundles. Here we try to optimize the performance of the method and examine the influence of certain parameters and modalities of the imaging on the quality and accuracy of the results.

The Use of Interlocking Prostheses for Both Temporary and Definitive Management of Infected Periprosthetic Femoral Fractures

Infected periprosthetic fractures around total hip arthroplasties are an extremely challenging problem. We describe our experience of managing infected periprosthetic femoral fractures using interlocking long-stem femoral prostheses either as temporary functional spacers or as definitive implants. The Cannulock (Orthodesign, Christchurch, United Kingdom) uncoated stem was used in 12 cases, and the Kent hip prosthesis (Biomet Merck, Bridgend, United Kingdom), in 5 cases. Satisfactory outcome was noted in all cases, and in 11 cases, revision to a definitive stem has been undertaken after successful control of infection and fracture union. The use of interlocking stems offers a relatively appealing solution for a complex problem and avoids the complications that would be associated with resection of the entire femur or the use of large quantities of bone cement.

Cold-neutron tomography of annular flow and functional spacer performance in a model of a boiling water reactor fuel rod bundle

Dryout of the coolant liquid film at the upper part of the fuel pins of a boiling water reactor (BWR) core constitutes the type of heat transfer crisis relevant for the conditions of high void fractions. It is both a safety concern and a limiting factor in the thermal power and thus for the economy of BWRs. We have investigated adiabatic, air–water annular flows in a scaled-up model of two neighboring subchannels as found in BWR fuel assemblies using cold-neutron tomography. The imaging of the double suchannel has been performed at the ICON beamline at the neutron spallation source SINQ at the Paul Scherrer Institute, Switzerland. Cold-neutron tomography is shown here to be an excellent tool for investigating air–water annular flows and the influence of functional spacers of different geometries on such flows. The high-resolution, high-contrast measurements provide the spatial distributions of the coolant liquid film thickness on the fuel pin surfaces as well as on the surfaces of the spacer vanes. The axial variations of the cross-section averaged liquid hold-up and its fraction in the gas core shows the effect of the spacers on the redistribution of the two phases.

Experimental studies of the effect of functional spacers to annular flow in subchannels of a BWR fuel element

For the prediction of dryout in fuel elements of boiling water reactors, the dynamic behavior of the water film covering the fuel rod has to be understood. This paper provides high resolved experimental data of the liquid film and gives insight into the dynamic film behavior. The experiments of this work were conducted in a vertical channel representing a pair of neighboring subchannels of a BWR fuel rod bundle. Air and water at ambient pressure and temperature are used as model fluids, creating an annular flow in the test section. The influence of different functional spacer shapes on the liquid film has been studied. The heart of the instrumentation is a liquid film sensor (LFS), which measures the film thickness distribution around a half cylinder with a matrix of 64 × 16 measuring points with a time resolution of 10,000 frames per second and a spatial resolution of 2 mm × 2 mm. The high resolution allows for a visualization of the dynamic liquid film as a movie animation. Principals of the dynamic behavior of the liquid film are observed. The time-averaged film thickness distributions show that the spacers structure the liquid film significantly. The gaseous phase is accelerated due to the cross-section blockage caused by the spacer. This leads to a local thinning of the liquid film downstream of the spacer. Two statistical evaluation methods are presented to determine different dynamic wave properties: The wave velocity as function of the wave height, the traveling path of the waves and the location of wave separation and merge events. The first evaluation method shows that big waves move generally faster than small waves. The analysis further shows wave acceleration in close proximity of the spacer with subsequent deceleration further downstream. Analyzing the wave as a two-dimensional entity it can be seen that the wave paths are clearly structured by the spacer and hence do not travel circumferentially around the fuel rod. Wave separation and merge has a higher occurrence probability in the subchannel gap, if there is no spacer present. With spacer the merge and separation locations are only found along the main flow paths of the waves.