Probe-target Complex Research Articles

A Dual-Fluorescence Molecular "Open Bridge" for Evaluating Gestational Hypoxia and Hypertension under the Stress of SARS-Cov-2.

Gestational hypertension is a dangerous condition that is sometimes fatal to the mother and her unborn off-spring. The strong connection between hypertension and hypoxia is emphasized by the currently rampaging SARS-Cov-2, which can induce similar conditions, in which hemolysis and the subsequent oxidative damage may release hemoglobin and tissue factor into the serum. To detect these dangerous proteins normally absent from serum, we mimic the molecular pathology of hypoxia, resulting in a synthesizable molecular machine around which a new bioassay can be designed to simultaneously detect the two proteins in a one-step and reagentless fashion. The "open bridge"-like probe can split into two upon ATP-induced cross-linking of hemoglobin to the probe. The covalently captured hemoglobin can subsequently use its peroxidase-like activity to induce a second cross-coupling between the probe and the tissue factor. A fluorescent probe-target covalent complex is formed, enabling thorough rinsing to minimize nonspecific interference. Finally, using hemoglobin's peroxidase activity to improve sensitivity, the assay has been successfully applied in detecting the two proteins in the periphery serum of pregnant women. These results may promise a near future application of the proposed method for providing an early warning for gestational hypoxia and hypertension, particularly under the stress of SARS-Cov-2.

A novel universal fluorescence detection platform based on target binding-mediated primer exchange reaction strategy for sensitive and accurate detection of multiple biomolecules

This study introduces a universal fluorescent detection platform based on the binding-mediated primer exchange reaction strategy, designed for highly sensitive and accurate detection of multiple biomolecules. This method uses an intact DNA hybridization probe as the initial target recognition site, forming a stable probe-target complex upon binding to the target. This complex drives the primer exchange reaction to amplify the signal, and then to achieve signal output through fluorescence resonance energy transfer (FRET). The reaction process is initiated directly by the catalytic target binding medium, ensuring method specificity. The versatile platform, equipped with various recognition elements such as antisense nucleic acids, biotin, and split aptamers, demonstrates exceptional universality in detecting a range of biomolecules, including virus nucleic acid fragments, streptavidin, and ATP. The method exhibits strong linear relationships with detection limits of 1.02 pM for virus nucleic acid fragments, 1.08 nM for streptavidin, and 15.7 μM for ATP, respectively. Moreover, the method displays excellent selectivity and outstanding performance in complex biological samples. This promising biosensing platform is believed to find numerous applications spanning bioanalysis, disease diagnosis, and biomedicine.

Photoluminescence study of SBA-15@ZnO/Au nanocomposite for potential use in Staphylococcus aureus detection

Staphylococcus aureus is most common causes of hospital-acquired infections and food-associated disease. In the last years, sensing platform based on fluorescence- nanomaterials and nanocomposites have been attracted a great attention for detection of bacteria and macromolecules. In this study, a new modified mesoporous silica SBA-15 with ZnO and Au nanoparticles (SBA-15@ZnO/Au) was prepared to enhance the photoluminescence emission of SBA-15 matrix and investigate the potential use as a nano probe for detection of S. aureus bacteria. Au nanoparticles were added to the SBA-15 after loading ZnO nanoparticles and the SBA-15@ZnO/Au nanocomposite was successfully fabricated. The Structural, morphological, physicochemical and optical properties of the prepared samples have been studied. The low-angle X-ray diffraction (XRD) pattern showed highly ordered two-dimensional hexagonal lattice of SBA-15 and the wide-angle XRD pattern proved the formation of ZnO and Au nanoparticles. The results of the Field emission scanning electron microscopy (FESEM) and transmission electron microscopy (TEM) analysis showed that the prepared nanocomposite have a regular two dimensional hexagonal structure with cylindrical channels even after loading ZnO and Au nanoparticles. According to Nitrogen adsorption–desorption isotherm measurement, the pore diameter and the specific surface area of SBA-15@ZnO/Au nanocomposite have been obtained 3.15 nm and 604.19 m2/g. Photoluminescence (PL) spectra of bare SBA-15@ZnO/Au nanocomposite showed three peaks at central wavelength of 410, 500 and 750 nm arising from electron transfer process and radiative decay of collective plasmonic states. After addition of S. aureus bacteria, the nano probe-target complex was formed and the PL re-emission at 640 nm and quenching of PL emissions at 750 and 410 nm was observed. Furthermore, the new nanocomposite was demonstrated to have the proper sensitivity toward bacteria concentrations. The results can open promising prospect for this nanocomposite as a nano probe for detection of S. aureus in the UV–Visible region of electromagnetic spectrum.

Pyrrolidinyl Peptide Nucleic Acid Probes Capable of Crosslinking with DNA: Effects of Terminal and Internal Modifications on Crosslink Efficiency.

In this study, we describe a furan-modified acpcPNA as a probe that can form an interstrand crosslink (ICL) with its DNA target upon activation with N-bromosuccinimide (NBS). To overcome the problem of furan instability under acidic conditions, a simple and versatile post-synthetic methodology for the attachment of the furan group to the PNA probe was developed. Unlike in other designs, the furan was placed at the end of the PNA molecule or tethered to the PNA backbone with all the base pairs in the PNA ⋅ DNA duplexes fully preserved. Hence, the true reactivity of each nucleobase towards the crosslinking could be compared. We show that all DNA bases except T could participate in the crosslinking reaction when the furan was placed at the end of the PNA strand. The crosslinking process was sensitive to mispairing, and lower crosslinking efficiency was observed in the presence of a base-mismatch in the PNA ⋅ DNA duplex. In contrast, when the furan was placed at internal positions of the acpcPNA ⋅ DNA duplex, no ICL was observed; this was explained by the inability of a hydrogen-bonded nucleobase to participate in the crosslinking reaction. The crosslinking efficiency was considerably improved, despite lower duplex stability, when an unpaired base (in the form of C-insertion) was present in the complementary DNA strand close to the furan modification site.

Generation and Single-Molecule Characterization of a Sequence-Selective Covalent Cross-Link Mediated by Mechlorethamine at a C–C Mismatch in Duplex DNA for Discrimination of a Disease-Relevant Single Nucleotide Polymorphism

Many strategies for the detection of nucleic acid sequence rely upon Watson-Crick hybridization of a probe strand to the target strand, but the reversible nature of nucleic acid hybridization presents an inherent challenge: short probes that provide high target specificity have relatively low target affinity resulting in signal losses. Sequence-specific covalent cross-linking reactions have the potential to provide both selective target capture and durable signal. We explore a novel approach involving sequence-specific covalent cross-linking of a probe to target DNA combined with single-molecule nanopore detection of the cross-linked DNA. Here, we exploited the selective reaction of mechlorethamine at a C-C mismatch for covalent capture of a target DNA sequence corresponding to a cancer-driving mutation at position 1799 of the human BRAF kinase gene. We then demonstrated that the α-hemolysin protein nanopore can be employed for the unambiguous, single-molecule detection of the cross-linked probe-target complex. Cross-linked DNA generates an unmistakable deep and persistent current block (≥5 s) that is easily distinguished from the microsecond and millisecond blocks generated by translocation of single-stranded DNA and uncross-linked duplexes through the nanopore.

Divide and Control: Comparison of Split and Switch Hybridization Sensors.

Hybridization probes have been intensively used for nucleic acid analysis in medicine, forensics and fundamental research. Instantaneous hybridization probes (IHPs) enable signalling immediately after binding to a targeted DNA or RNA sequences without the need to isolate the probe-target complex (e. g. by gel electrophoresis). The two most common strategies for IHP design are conformational switches and split approach. A conformational switch changes its conformation and produces signal upon hybridization to a target. Split approach uses two (or more) strands that independently or semi independently bind the target and produce an output signal only if all components associate. Here, we compared the performance of split vs switch designs for deoxyribozyme (Dz) hybridization probes under optimal conditions for each of them. The split design was represented by binary Dz (BiDz) probes; while catalytic molecular beacon (CMB) probes represented the switch design. It was found that BiDz were significantly more selective than CMBs in recognition of single base substitution. CMBs produced high background signal when operated at 55°C. An important advantage of BiDz over CMB is more straightforward design and simplicity of assay optimization.

Enzyme-assisted target recycling (EATR) for nucleic acid detection.

Fast, reliable and sensitive methods for nucleic acid detection are of growing practical interest with respect to molecular diagnostics of cancer, infectious and genetic diseases. Currently, PCR-based and other target amplification strategies are most extensively used in practice. At the same time, such assays have limitations that can be overcome by alternative approaches. There is a recent explosion in the design of methods that amplify the signal produced by a nucleic acid target, without changing its copy number. This review aims at systematization and critical analysis of the enzyme-assisted target recycling (EATR) signal amplification technique. The approach uses nucleases to recognize and cleave the probe-target complex. Cleavage reactions produce a detectable signal. The advantages of such techniques are potentially low sensitivity to contamination and lack of the requirement of a thermal cycler. Nucleases used for EATR include sequence-dependent restriction or nicking endonucleases or sequence independent exonuclease III, lambda exonuclease, RNase H, RNase HII, AP endonuclease, duplex-specific nuclease, DNase I, or T7 exonuclease. EATR-based assays are potentially useful for point-of-care diagnostics, single nucleotide polymorphisms genotyping and microRNA analysis. Specificity, limit of detection and the potential impact of EATR strategies on molecular diagnostics are discussed.

Isotachophoresis with ionic spacer and two-stage separation for high sensitivity DNA hybridization assay

We present an on-chip electrophoretic assay for rapid and high sensitivity nucleic acid (NA) detection. The assay uses isotachophoresis (ITP) to enhance NA hybridization and an ionic spacer molecule to subsequently separate reaction products. In the first stage, the probe and target focus and mix rapidly in free solution under ITP. The reaction mixture then enters a region containing a sieving matrix, which allows the spacer ion to overtake and separate the slower probe-target complex from free, unhybridized probes. This results in the formation of two focused ITP peaks corresponding to probe and probe-target complex signals. For a 149 nt DNA target, we achieve a 220 fM limit of detection (LOD) within 10 min, with a 3.5 decade dynamic range. This LOD constitutes a 12× improvement over previous ITP-based hybridization assays. The technique offers an alternative to traditional DNA hybridization assays, and can be multiplexed and extended to detect other biomolecules.

A Simple Method for Amplifying RNA Targets (SMART)

We present a novel and simple method for amplifying RNA targets (named by its acronym, SMART), and for detection, using engineered amplification probes that overcome existing limitations of current RNA-based technologies. This system amplifies and detects optimal engineered ssDNA probes that hybridize to target RNA. The amplifiable probe-target RNA complex is captured on magnetic beads using a sequence-specific capture probe and is separated from unbound probe using a novel microfluidic technique. Hybridization sequences are not constrained as they are in conventional target-amplification reactions such as nucleic acid sequence amplification (NASBA). Our engineered ssDNA probe was amplified both off-chip and in a microchip reservoir at the end of the separation microchannel using isothermal NASBA. Optimal solution conditions for ssDNA amplification were investigated. Although KCl and MgCl(2) are typically found in NASBA reactions, replacing 70 mmol/L of the 82 mmol/L total chloride ions with acetate resulted in optimal reaction conditions, particularly for low but clinically relevant probe concentrations (≤100 fmol/L). With the optimal probe design and solution conditions, we also successfully removed the initial heating step of NASBA, thus achieving a true isothermal reaction. The SMART assay using a synthetic model influenza DNA target sequence served as a fundamental demonstration of the efficacy of the capture and microfluidic separation system, thus bridging our system to a clinically relevant detection problem.

General Colorimetric Detection of Proteins and Small Molecules Based on Cyclic Enzymatic Signal Amplification and Hairpin Aptamer Probe

In this work, we developed a simple and general method for highly sensitive detection of proteins and small molecules based on cyclic enzymatic signal amplification (CESA) and hairpin aptamer probe. Our detection system consists of a hairpin aptamer probe, a linker DNA, two sets of DNA-modified AuNPs, and nicking endonuclease (NEase). In the absence of a target, the hairpin aptamer probe and linker DNA can stably coexist in solution. Then, the linker DNA can assemble two sets of DNA-modified AuNPs, inducing the aggregation of AuNPs. However, in the presence of a target, the hairpin structure of aptamer probe is opened upon interaction with the target to form an aptamer probe-target complex. Then, the probe-target complex can hybridize to the linker DNA. Upon formation of the duplex, the NEase recognizes specific nucleotide sequence and cleaves the linker DNA into two fragments. After nicking, the released probe-target complex can hybridize with another intact linker DNA and the cycle starts anew. The cleaved fragments of linker DNA are not able to assemble two sets of DNA-modified AuNPs, thus a red color of separated AuNPs can be observed. Taking advantage of the AuNPs-based sensing technique, we are able to assay the target simply by UV-vis spectroscopy and even by the naked eye. Herein, we can detect the human thrombin with a detection limit of 50 pM and adenosine triphosphate (ATP) with a detection limit of 100 nM by the naked eye. This sensitivity is about 3 orders of magnitude higher than that of traditional AuNPs-based methods without amplification. In addition, this method is general since there is no requirement of the NEase recognition site in the aptamer sequence. Furthermore, we proved that the proposed method is capable of detecting the target in complicated biological samples.

Dual fluorophore PNA FIT-probes − extremely responsive and bright hybridization probes for the sensitive detection of DNA and RNA

Fluorescently labeled oligonucleotides are commonly employed as probes to detect specific DNA or RNA sequences in homogeneous solution. Useful probes should experience strong increases in fluorescent emission upon hybridization with the target. We developed dual labeled peptide nucleic acid probes, which signal the presence of complementary DNA or RNA by up to 450-fold enhancements of fluorescence intensity. This enabled the very sensitive detection of a DNA target (40 pM LOD), which was detectable at less than 0.1% of the beacon concentration. In contrast to existing DNA-based molecular beacons, this PNA-based method does not require a stem sequence to enforce dye-dye communication. Rather, the method relies on the energy transfer between a "smart" thiazole orange (TO) nucleotide, which requires formation of the probe-target complex in order to become fluorescent, and terminally appended acceptor dyes. To improve upon fluorescence responsiveness the energy pathways were dissected. Hydrophobic, spectrally mismatched dye combinations allowed significant (99.97%) decreases of background emission in the absence of a target. By contrast, spectral overlap between TO donor emission and acceptor excitation enabled extremely bright FRET signals. This and the large apparent Stokes shift (82 nm) suggests potential applications in the detection of specific RNA targets in biogenic matrices without the need of sample pre-processing prior to detection.

A highly efficient and versatile microchip capillary electrophoresis method for DNA separation using gold nanoparticle as a tag

A highly efficient and versatile method for DNA separation using Au nanoparticles (Au NPs) as a tag based on microchip capillary electrophoresis (MCE) was developed. The thiol-modified DNA-binding Au NPs were utilized as a tag. Target DNA was sandwiched between Au NPs and probe DNA labeled with horseradish peroxidase (HRP). In electrophoresis separation, the difference in electrophoretic mobility between free probe and probe-target complex was magnified by Au NPs, which enabled the resulting mixture to be separated with high efficiency by microchip capillary electrophoresis. Horseradish peroxidase was used as a catalytic label to achieve sensitive electrochemical DNA detection via fast catalytic reactions. With this protocol, 27-mer DNA fragments with different sequences were separated with high speed and high resolution. The proposed method was critical to achieve improved DNA separations in hybridization analyses.

Designed thiazole orange nucleotides for the synthesis of single labelled oligonucleotides that fluoresce upon matched hybridization

Probe molecules that enable the detection of specific DNA sequences are used in diagnostic and basic research. Most methods rely on the specificity of hybridization reactions, which complicates the detection of single base mutations at low temperature. Significant efforts have been devoted to the development of oligonucleotides that allow discrimination of single base mutations at temperatures where both the match and the mismatch probe-target complexes coexist. Oligonucleotides that contain environmentally sensitive fluorescence dyes such as thiazole orange (TO) provide single nucleotide specific fluorescence. However, most previously reported dye-DNA conjugates showed only little if any difference between the fluorescence of the single and the double stranded state. Here, we introduce a TO-containing acyclic nucleotide, which is coupled during automated oligonucleotide synthesis and provides for the desired fluorescence-up properties. The study reveals the conjugation mode as the most important issue. We show a design that leads to low fluorescence of the unbound probe (background) yet permits TO to adopt fluorescent binding modes after the probe-target complex has formed. In these probes, TO replaces a canonical nucleobase. Of note, the fluorescence of the "TO-base" remains low when a base mismatch is positioned in immediate vicinity.

Detection of single nucleotide polymorphisms using a DNA Holliday junction nanoswitch—a high-throughput fluorescence lifetime assay

We report a simple DNA sensor device, using a combination of binding and conformational switching, capable of rapid detection of specific single nucleotide polymorphisms in an unlabelled nucleic acid target sequence. The detection is demonstrated using fluorescence lifetime measurements in a high-throughput micro plate reader instrument based on the time-correlated single-photon counting technique. The sensor design and instrumental architecture are capable of detecting perturbations in the molecular structure of the probe-target complex (which is similar to that of a Holliday junction), due to a single base pair mismatch in a synthetic target. Structural information, including fluorophore separations, is obtained using time-resolved Förster resonance energy transfer between two fluorophores covalently bound to the probe molecule. The two probes required are designed to detect a single nucleotide polymorphism from a sequence present on each of the two copies of human chromosome 11.

Electrochemical control of a DNA Holliday Junction nanoswitch by Mg 2+ ions

The molecular conformation of a synthetic branched, 4-way DNA Holliday junction (HJ) was electrochemically switched between the open and closed (stacked) conformers. Switching was achieved by electrochemically induced quantitative release of Mg 2+ ions from the oxidised poly( N-methylpyrrole) film (PPy), which contained polyacrylate as an immobile counter anion and Mg 2+ ions as charge compensating mobile cations. This increase in the Mg 2+ concentration screened the electrostatic repulsion between the widely separated arms in the open HJ configuration, inducing switching to the closed conformation. Upon electrochemical reduction of PPy, entrapment of Mg 2+ ions back into the PPy film induced the reverse HJ switching from the closed to open state. The conformational transition was monitored using fluorescence resonance energy transfer (FRET) between donor and acceptor dyes each located at the terminus of one of the arms. The demonstrated electrochemical control of the conformation of the used probe-target HJ complex, previously reported as a highly sequence specific nanodevice for detecting of unlabelled target [Buck, A.H., Campbell, C.J., Dickinson, P., Mountford, C.P., Stoquert, H.C., Terry, J.G., Evans, S.A.G., Keane, L., Su, T.J., Mount, A.R., Walton, A.J., Beattie, J.S., Crain, J., Ghazal, P., 2007. Anal. Chem., 79, 4724–4728], allows the development of electronically addressable DNA nanodevices and label-free gene detection assays.

Theoretical and Experimental Analysis of Arrayed Imaging Reflectometry as a Sensitive Proteomics Technique

Arrayed imaging reflectometry (AIR) is a newly developed label-free optical biosensing technique based on the creation and perturbation of a condition of zero reflectance on a silicon substrate. The antireflective coating is formed by covalently immobilizing arrayed probes on a silicon dioxide film. Probe-target complex formation causes a localized increase in optical thickness and a measurable reflectance change. To evaluate the performance of AIR, we have employed two proteins, intimin and tir, from enteropathogenic E. coli that are critical to the bacterium's mechanism of host infection. Using substrates functionalized with the intimin-binding domain of tir, we demonstrate detection of the extracellular domain of intimin at concentrations as low as 10 pM. Through the use of a diffusion-limited model for the intimin-tir binding interaction at this concentration, we estimate the detected intimin surface concentration to be 0.33 pg/mm2.

Cover Picture: Forced Intercalation Probes (FIT Probes): Thiazole Orange as a Fluorescent Base in Peptide Nucleic Acids for Homogeneous Single‐Nucleotide‐Polymorphism Detection (ChemBioChem 1/2005)

The cover picture shows new PNA probes–FIT Probes–and their use in homogenous DNA detection and was designed by Elke Socher. In FIT Probes, the thiazole orange dye is forced to intercalate at a specific position of a probe–target complex. In their paper on p. 69 ff, O. Seitz et al. describe how the binding of FIT probes with DNA results in fluorescence enhancements due to the coplanarization of the two heterocyclic ring systems. The remarkable attenuation of fluorescence that is observed when forcing thiazole orange to intercalate next to a mismatched base pair allows a DNA target to be distinguished from its single‐base mutant under nonstringent hybridization conditions, a property that should be of advantage for real‐time quantitative PCR and nucleic acid detection within living cells.

Direct quantification of mRNA expression levels using single molecule detection

Determination of the gene expression by direct quantification of mRNA is becoming increasingly important in basic, pharmaceutical and clinical research. We present a novel approach for gene quantification based on direct hybridization of gene-specific probes to target mRNA sequences in solution at temperatures ensuring absolute specificity of the probe-target complex. No enzymatic steps like reverse transcription or amplification by PCR are involved within the quantification process. In order to increase specificity as well as sensitivity, two probes emitting fluorescence light in different colors are used for our homogeneous assay using fluorescence cross-correlation. We relate to the single molecule sensitivity of excitation and detection in confocal cavities avoiding the amplification of the detected signal. The analysis of the expression level of high, medium and low abundant genes is described in two different cell lines, whereby the genes are quantified in absolute numbers.

Using DNA-binding proteins as an analytical tool.

We propose that DNA-binding proteins can be used as highly efficient and versatile tools in analyses of DNA, RNA, and proteins. This work reports assays applying specific affinity probes: hybridization probes for analyses of DNA and RNA, and aptamer probes for analyses of proteins. Both types of probes are single-stranded DNA. In affinity analyses, in general, the probe (P) binds to a target molecule (T), and the amounts of the probe-target complex (P.T) and unbound P are determined. Distinguishing between P and P.T can be achieved by electrophoretic separation. If the electrophoretic mobilities of P and P.T are close in gel-free media, which is always the case for hybridization analyses, separation typically requires the use of a sieving matrix. Here we utilized a single-stranded DNA binding protein (SSB) to facilitate highly efficient gel-free separation of P and P.T in capillary electrophoresis (CE) for three types of targets: DNA, RNA, and proteins. When present in the CE run buffer, SSB binds differently to P and P.T. Due to this selective binding, SSB induces difference in electrophoretic mobilities of P and P.T in an SSB concentration-dependent fashion. The difference in the electrophoretic mobilities allows for affinity analyses of DNA, RNA, and proteins in gel-free CE. The large number of well-characterized DNA- and RNA-binding proteins and the diversity of their properties will allow researchers to design a comprehensive tool set for quantitative analyses of DNA, RNA, and proteins. Such analyses will facilitate identification of genomic DNA in ultra-small samples without error-prone and time-consuming PCR. They can also be used for monitoring gene expression at both mRNA and protein levels.

Automated kappa and lambda light chain mRNA expression for the assessment of B-cell clonality in cutaneous B-cell infiltrates: its utility and diagnostic application.

Primary cutaneous B-cell lymphoma (1 degrees CBCL) accounts for 25% of all lymphomas. The difficulty in distinction of reactive from neoplastic B-cell infiltrates prompts the use of molecular diagnostic adjuncts. While T-cell clonality can be seen in various reactive states, clonal B-cell infiltrates are often neoplastic; standard assays employed include polymerase chain reaction (PCR) or Southern blot analysis to assess heavy chain rearrangement. We sought to assess the utility of kappa (kappa) and lambda (lambda) mRNA expression using the Ventana automated assay (Ventana Medical Systems, Tucson, AZ, USA) in the analysis of atypical cutaneous B-cell lymphoid infiltrates. Multiple 4 micro m sections of paraffin-embedded, formalin-fixed skin biopsies from 31 patients with CBCL were placed on silane-coated slides, deparaffinized, then digested in pepsin (5 mg/ml) for 30 min at 37 degrees C. Fluorescein-tagged oligoprobes and tissue mRNA were denatured at 80 degrees C for 5 min, hybridized for 2 h at 37 degrees C, and incubated with antifluorescein alkaline phosphatase conjugates. Detection of the probe target complex employed nitroblue tetrazolium and bromochloroindolyl phosphate conjugates with a nuclear fast red counterstain. A kappa : lambda ratio > 3 : 1 was held to represent kappa light chain restriction and a kappa : lambda ratio </= 1 : 1 to indicate lambda light chain restriction. The diagnosis in each case was determined by careful integration of clinical, histologic, and phenotypic data. The diagnoses included: pseudolymphoma (PL), marginal zone lymphoma (MZL), 1 degrees CBCL of the trunk, scalp or leg, 2 degrees lymphoma, and plasma cell dyscrasia. All but one case of lymphoma were light chain restricted. All cases of PL were proven to be polyclonal by this methodology. In non-plasmacytic small cell lymphomas, only 5-10% of the infiltrate expressed kappa or lambda, with clonality established through the abnormal kappa : lambda ratio. Interpretations were most difficult in the 2 degrees small cell-dominant follicular center cell lymphomas and easiest in cases with significant plasmacytic differentiation (i.e. MZL, immunocytomas, or plasma cell dyscrasias). The Ventana kappa/lambda assay is a reliable, quick, and inexpensive way to determine B-cell clonality in cutaneous lymphoid infiltrates in paraffin-embedded formalin-fixed tissue.