Medicinal plants have been used by humans since ancient times for the treatment of various diseases and currently represent the main source of a variety of phytocompounds, such as triterpenes. Pentacyclic triterpenes have been subjected to numerous studies that have revealed various biological activities, such as anticancer, antidiabetic, anti-inflammatory, antimicrobial, and hepatoprotective effects, which can be employed in therapy. However, due to their high lipophilicity, which is considered to exert a significant influence on their bioavailability, their current use is limited. A frequent approach employed to overcome this obstacle is the chemical derivatization of the core structure with different types of moieties including heterocycles, which are considered key elements in medicinal chemistry. The present review aims to summarize the literature published in the last 10 years regarding the derivatives of pentacyclic triterpenes bearing heterocyclic moieties and focuses on the biologically active derivatives as well as their structure–activity relationships. Predominantly, the targeted positions for the derivatization of the triterpene skeleton are C-3 (hydroxyl/oxo group), C-28 (hydroxyl/carboxyl group), and C-30  or the extension of the main scaffold by fusing various heterocycles with the A-ring of the phytocompound. In addition, numerous derivatives also contain linker moieties that connect the triterpenic scaffold with heterocycles; one such linker, the triazole moiety, stands out as a key pharmacophore for its biological effect. All these studies support the hypothesis that triterpenoid conjugates with heterocyclic moieties may represent promising candidates for future clinical trials. Since ancient times, humans have used herbs to cure various diseases, and even though they were initially instinctively chosen, people began to know precisely which plants to use for a particular disease due to the experience gained over time . NowadayPentacyclic triterpenes are a well-represented class of phytocompounds resulting from plants’ secondary metabolism, mainly through squalene cyclization ; they cahttp://www.ncbi.nlm.nih.gov/pubmed, accessed on 13 May 2022) and Web of Science . The databases were searched using combinations of the key terms “pentacyclic triterpenes” and “heterocycle”, covering the last 10 years. The inclusion criteria for the current manuscript included: (1) original articles; (2) abstracts or full text available; (3) articles containing data about pentacyclic triterpene heterocyclic derivatives and bioconjugates. The exclusion criteria included: (1) duplicates; (2) articles in languages other than English; (3) review-type papers; (4) irrelevant articles for the topic; (5) articles that discussed only the synthesis of the derivatives without their biological assessment; (6) articles that discussed ring-modified derivatives. Regarding the studies that focused on obtaining not only heterocyclic derivatives but also aryl-derivatives, the review also presents these aryl-derivatives due to the fact that they exhibited significant biological activities in their respective studies, even if they could not be classified as heterocyclic derivatives or bioconjugates. The database screening resulted in a total of 143 articles; however, after the application of the above-mentioned inclusion and exclusion criteria, only 90 articles were subjected to further analysis.The current work aims to summarize the most recent published studies on the topic of semisynthetic derivatives of pentacyclic triterpenes bearing heterocyclic moieties and focuses on biologically active derivatives and their structure–activity relationships (SAR). The studies cited were collected from two major databases recognized by the scientific community, PubMed (Lupeol (lup-20(29)-en-3β-ol; Khan et al. obtained a series of C19-isopropylene-modified lupeol derivatives and further assessed their influence on in vitro glucose absorption. The results obtained for several heterocyclic derivatives either containing a pyrazoline moiety Lup 1–3;  or obtai50 > 70 µM, a Lupeol–C16-sulfate derivative, as well as with doxorubicin used as a standard drug [A series of lupeol derivatives was synthesized by Castro et al. through C3, C16, or C-19-isopropylene chemical modulation, followed by their cytotoxicity assessment against two prostate cancer cell lines, LNCaP and PC-3. Among the tested derivatives, two compounds bearing a double esterification at the C3- and C-16 hydroxyl groups with benzoyl chloride (Lup 12) or p-bromo-benzoyl chloride (Lup 13), respectively, exerted lower cytotoxicity compared with the lead compound ICard drug . The autard drug ; howeverBetula alba, B. pubescens, B. pendula) [Betulin (lup-20(29)-ene-3β,28-diol, betulinol) is a lupane-type pentacyclic triterpenoid bearing two C3, C28-hydroxyle, and one isopropylene moiety at C19 ; this phpendula) . Betulinpendula) . In ordependula) . Saponins represent a heterogeneous class of triterpenoids or steroid glycosides that are widely distributed in nature and exhibit various biological activities, including not only antiproliferative effects against many types of cancers but also antiviral, antifungal, and anti-inflammatory activities . HoweverTomanek et al. tested the biological benefits of 1,4-quinone-conjugated betulin against seven cancer cell lines. Data showed that the quinone moiety attached to betulin at C-28 through a triazole linker was essential for exhibiting anticancer activity; such compounds exerted the strongest anticancer activity against lung cancer (A549 cell line). SAR analysis revealed that the position of the nitrogen atom plays a significant role in terms of future biological effects; thus, compounds (Bet 2–5) that contain a 5,8-isoquinolinedione group exhibited higher activity than those containing a 5,8-quinolinedione moiety. Furthermore, the particular structure of the 1,4-quinone scaffold strongly influences the potency of the biological activity in the following sequence: 2-methyl-5,8-quinolinedione > 1,4-naphtoquinone > 5,8-isoquinolinedione > 5,8-quinolinedione [The triazole linker was also used by Anh et al., who synthesized a series of eleven compounds through click chemistry by coupling C28-betulin-triazoles with the antiviral 3′-azido-3′deoxythydimine (AZT); the compounds were further tested in vitro against KB  and HepG2 (liver carcinoma) cancer cell lines. The results showed that all compounds exerted at least moderate cytotoxic activity against both cancer cell lines; in particular, the hybrid Bet 6 , containThe assessment of betulin bioconjugates in terms of their effects on the mitochondrial membrane potential and, subsequently, on the oxidative phosphorylation was conducted by Dubinin et al., who synthesized a novel betulin conjugate by attaching the (E)-4-(1h Indol-3-yl-vinyl)pyridine fragment to the C-28 bromoalkyl betulin ester (Bet 7) and evaluated its cytotoxic activity in isolated rat mitochondria. The authors previously demonstrated that betulin can solely modify the mitochondrial membrane; in addition, the newly developed betulin conjugate exhibited a strong cytotoxic effect by selectively accumulating in mitochondria, thus leading to the suppression of respiration and oxidative phosphorylation as well as ROS overexpression, finally resulting in cell death . Leishmania infantum growth; by contrast, the addition of carbamate moieties in the same position provided lower inhibitory effects compared with the parent compound. The most active compound against the promastigote forms of L. infantum was 3-hydroxy-20-lupan-29-oxo-yl-1H-imidazole-1-carboxylate (Bet 8), having its C-20-29 double bond oxidized into an aldehyde and displaying an imidazole scaffold grafted at the C-28 position through an ester bond, which revealed an IC50 value of 50.8 μM; furthermore, when administered in combination with miltefosine, the same betulin derivative exerted a synergistic effect, with the IC50 value decreasing to 6 μM. Most importantly, the compound did not cause any significant macrophage necrosis, thus suggesting little to no toxicity to the host cell [Betulin imidazole derivatives have been tested against leishmaniasis, a highly difficult-to-treat tropical disease; Sousa et al. obtained a series of nine betulin derivatives that were further assessed in terms of pharmacological interaction with miltefosine, an antileishmanial drug currently used in polytherapy. According to SAR analysis, the insertion of imidazole moieties at the C-28 position through an ester bond induced highly promising inhibitory effects against ost cell . 50 values within the micromolar range were calculated; moreover, the selected compounds proved to be harmless against healthy human fibroblasts [The insertion of an isoxazole moiety at C-28 through the oxidation of the hydroxyl group followed by condensation and cycloaddition led to a series of compounds that were further tested against MCF7 breast cancer, U-87 MG multiforme glioblastoma, A549 lung carcinoma, and HepG2 hepatocarcinoma cell lines, as well as in hTERT human fibroblasts. The SAR study proved that the substituents attached to the isoxazole ring significantly influenced the overall cytotoxic effects; as an example, the saponin-like derivatives containing glucose substituents grafted on the isoxazole fragment induced cytotoxic effects against A549 and MCF7 cell lines, but no activity was recorded in the HepG2 cells. The most promising results were reported for derivatives bearing small hydrophilic substituents on the isoxazole ring, such as hydroxymethyl (Bet 9–10), 2-hydroxypropan-2-yl (Bet 11), (2-hydroxyethoxy)methyl (Bet 12), carboxyl (Bet 13), and (acetylthio)methyl (Bet-14), for which ICroblasts . P. falciparum malaria parasite. A series of hybrid derivatives were synthesized and analyzed in vitro against malaria parasites as well as human cytomegalovirus (HCMV). The hybrids were obtained with several esterification reactions, either between individual molecules of betulin and artesunic acid or by additionally using ferrocene moieties as linkers. The most active compound was Bet 15, obtained using C28-betulin esterification with one molecule of artesunic acid, which exhibited a 20-fold activity increase compared to botulin, as indicated by the EC50 value of 0.24 μM. However, compared with the effect of chloroquine against P. falciparum, none of the derivatives presented significantly improved antimalarial activity [Betulin and artesunic acid were chosen for the development of new molecules that could effectively treat the drug-resistant activity . E. coli), and eukaryotic cells (rat thymocytes), as well as on rat liver mitochondria. The study showed that all three compounds inhibited the growth of E. coli without exerting a cytotoxic effect on rat thymocytes. However, the presence of the methyl substituents on the pyridine ring was essential for their activity in mitochondria, with a SAR assessment proving that 30-pyridinium-1-yl-lup-20(29)-ene-3,28-diol bromide, which lacks methyl groups, was not able to induce mitochondrial swelling and aggregation; hence, it did not cause membrane permeabilization. Moreover, Bet 16  and S. aureus (minimum inhibitory concentration of 1 μg/mL) [Similarly, three derivatives were synthesized using pyridinium fragments; the conjugates were obtained by attaching the pyridinium fragment to the C-30 allylic bond of betulin, betulonic acid, and betulin 28-O-acetate, leading to the increased polarity of the molecule and creating new amphiphilic betulin conjugates. The antibacterial and antifungal properties of the three derivatives and their triterpenic analogs were tested against both Gram-positive 1 μg/mL) The previously mentioned C28 modulation by using 1,4-quinone moieties can also be conducted in other positions on the betulin scaffold; a large series of betulin-1,4-quinone hybrids were synthesized by conjugating betulin with various compounds bearing the 1,4-quinone fragment through the use of a linker. Different 1,4-quinone fragments were attached to C-3, C-28, and C-30 positions, respectively, of either betulin or betulin derivatives, such as 3-acetylbetulin, 28-acetylbetulin, 28-propionylbetulin, and 30-hydroxy-3,28-diacetylbetulin. All derivatives were tested against a panel of human cancer cell lines, revealing that, in several compounds, the presence of the betulinyloxy group connected to the 1,4-quinone fragment increases their anticancer activity . Furthermore, compound Bet 28, containing a 30-deoxythymidine-5′-yl moiety fragment at C-28, was the only one with antibacterial activity against the two Gram- bacterial strains, K. pneumoniae and E. coli [The neuroprotective activity of several triterpenoid derivatives containing various sugar moieties was tested in vitro on neuron-like SH-SY5Y neuroblastoma cells; the compounds were prepared by attaching triazole-modified sugar moieties through ester bonds to the C30 position in the molecule of C3, C28-diacetylated betulin. The most efficient neuroprotective compound proved to be one betulin conjugate containing a tetraacetyl-β-glucose moiety Bet 23; ; further E. coli .50 = 0.66 μM) by inducing cycle arrest in the G2/M phase. Furthermore, a structure–activity relationship study on the series of the 19 derivatives suggested that the preferential integration of the triphenylphosphonium moiety into the C28 position might optimize the overall cytotoxic effect [The simultaneous modification of both the C-3 and C-28 positions in the betulin molecule may lead to the synthesis of novel mitochondria-targeted derivatives; such derivatives were obtained by attaching triphenylphosphonium moieties to the C-3 and/or C-28 acylated hydroxylic groups, resulting in nineteen betulin conjugates, which have been tested against four cancer cell lines, including human lung carcinoma (A549), human primary glioblastoma (U87), human cervical carcinoma (HeLa), and human colorectal carcinoma (HCT116), as well as one normal colon epithelial cell line (NCM460). After biological assessment, the most promising compound, Bet 29 , which wc effect . 50 of 0.045 μM. A SAR evaluation revealed that the triphenylphosphonium-substituted compounds induced stronger biological activities than both the parent compound and its acylated derivatives. Furthermore, the length of the alkyl chain linked to the ester bond significantly influenced the cytotoxic effect; as such, the methylene group induced lower biological activity compared with propylene and butylene fragments [The impact of grafting triphenylphosphonium moieties to the betulin scaffold was also investigated by Tsepaeva et al.; in a similar manner to the previous study, the triphenylphosphonium moiety was inserted into both the C-28 and C-3 positions, or into the C-28 position alone, through an acyl linker. The anticancer effects of the resulting compounds were assessed against several cancer cell lines: human breast cancer (MCF-7), prostate adenocarcinoma (PC-3), and vinblastine-resistant human breast cancer (MCF-7/Vinb), as well as on human skin fibroblast (HSF). Although most disubstituted compounds exhibited nanomolar range IC50 values, the most active derivative, Bet 30, possessing only one triphenylphosphonium moiety grafted at C-28, exerted superior cytotoxic activity against all tested cancer cell lines, but particularly against MCF-7/Vinb, with an ICragments . Considering the key role heterocycles play as pharmacophores in medicinal chemistry, Csuk et al. synthesized 24 betulin-derived propargylamines by first subjecting betulin to a series of transformations in order to obtain 28-propargyl betulins, which were then further conjugated with different types of N-bearing moieties through Mannich reactions using alkynes, aldehydes, and amines. The C-3 hydroxyl function was altered in some derivatives; hence, several hybrids were betulone derivatives as a result of oxidation, while others had their hydroxyl group previously acetylated. The isopropenyl moiety of the lupane skeleton remained unaffected for all derivatives. The betulin conjugates were further analyzed in terms of anticancer activity against nine human cancer cell lines, allowing for the formulation of some conclusions regarding their structure–activity relationships. A SAR analysis emphasized the importance of the C-28 alkynic function in the biological effect reported for the synthesized compounds. Furthermore, the introduction of N-heterocyclic substituents and amino moieties on the triterpenic scaffold led to significantly improved cytotoxic effects, while 3-O-acetyl hybrids exhibited higher biological activities compared to their deacetylated parent analogs . In order to evaluate the mechanism of mitochondrial toxicity, a series of dimethylaminopyridine derivatives with triterpenoid scaffolds were synthesized by Bernardo et al. by attaching one, two, or three pyridinium moieties to C-3, C-28, or C-30, respectively, in the molecule of betulin (four compounds) or betulinic acid (one compound); the five derivatives were selected from a larger series of previously synthesized compounds  due to s50 = 1.74µM) while moderately acting against normal cells, thus exhibiting some degree of selectivity. In terms of antimicrobial properties, all three betulin-3-yl glycosaminosides have poor-to-no activity against fungi as well as Gram-positive and Gram-negative bacteria [Another approach for the synthesis of novel betulin derivatives consisted of the introduction of 2-amino-2-deoxy-D-glucopyranosyl and -D-galactopyranosyl moieties, respectively, to the C30 position through a glycosidic bond. The resulting compounds, 28-acetylbetulin-3-yl-2-amino-2-deoxy-D-glucopyranoside and betulin-3-yl-2-amino-2-deoxy-D-gluco- and D-galactopyranoside (Bet 34–36), were further tested against the MCF-7 breast cancer cell line and HDFa human dermal fibroblasts, as well as against human pathogenic bacteria and fungi. The three compounds exhibited highly similar structures, the main differences consisting of the orientation of the hydroxyl groups in the sugar fragment and the presence/absence of C28-acetyl esterification. The experimental data indicated that all three saponins induced cytotoxic activities in both normal and cancer cells; however, betulin-3-yl-D-galactosamine (Bet 36) was the most active compound against MCF-7 cells  cells, which showed the highest sensitivity to the newly synthesized compounds [Numerous derivatives were obtained through modifications of ring A; Grishko et al. synthesized a novel series of A-ring-fused heterocyclic betulone derivatives, which were further tested against five cancer cell lines . According to a SAR study, the C(2)-C(3) fusion with oxazole, isoxazole, triazole, and triazine heterocycles, combined with a 28-hydroxyl moiety, induced far superior cytotoxic activities compared with their 28-acylated azole precursors. Moreover, the oxidation of the 28-hydroxyle moiety in the triazole-fused derivative led to significant suppression of the biological activity, while the presence of a 28-carboxyl fragment combined with a triazine moiety attached to the triterpenoid skeleton highly improved the cytotoxic effect. The study identified N-acetyltriazole betulin as a lead compound (Bet 41) due to its strong antiproliferative effect against all tested cancer cell lines, but particularly against HCT 116  and other inflammation markers. According to a SAR, the conjugates obtained by fusing a heterocyclic group to ring A exerted the highest inhibitory activity, especially when pyridine or pyrazine moieties were used. Moreover, the derivatization of the C-28 hydroxymethyl moiety into an oxime group significantly improved the compound’s biological activity; by contrast, the indole derivatives demonstrated weaker activity compared with the parent compound. The study revealed that compound Bet 42 (pyrazole A-fused betulin derivative), having an oxidized C-28 hydroxyl function, exhibited the highest in vitro inhibitory effect on carrageenan-induced paw inflammation in mice, as indicated by the reduction in nitric oxide production as well as the expression of pro-inflammatory cytokines and prostaglandin synthase-2 (COX-2) .Betulinic acid -en-28-oic acid; 50 = 7.80 nM [Wang et al. synthesized a BA-nucleoside bioconjugate (BA 1) through a click chemistry reaction between the C2-propargyl derivative of BA and 4′-azido-2′-deoxy-2′-fluoro-β-D-arabinocytidine—which exerted potent anti-HIV activity in vitro compared with the parent compounds—with an IC 7.80 nM . Further 7.80 nM . Altoget50 values of 1.62 µM and 1.19 µM, respectively. The SAR evaluation revealed that the introduction of aromatic N-heterocycles (pyrazine and pyridine) is unfavorable for the cytotoxic effect of the resulting derivative. In contrast, the introduction of saturated N-heterocycles (pyrrolidine and piperidine) significantly increases cytotoxicity, particularly against the multidrug-resistant cell line MCF-7/ADR [50 values ranging from 0.5 to 2.4 μM. A SAR evaluation also revealed that, in the series of C3-picolyl-BA derivatives (BA 4–6), the o- and m-substitution of the pyridine heterocycle favors cytotoxic activity, while in the series of C28-picolyl derivatives (BA 7–9), the p- and m-substitutions increase their cytotoxicity as opposed to the o-substitution [Several nitrogen-containing heterocycles were conjugated with BA by reacting the triterpenic acid and C3-amino-BA with various acids bearing pyrrolidine, piperidine, pyrazine, and pyridine moieties; all compounds were tested in terms of cytotoxicity against a panel of eight non-drug-resistant , one multidrug-resistant (MCF-7/ADR), and one normal  cell lines. The tests revealed two amide derivatives, compounds BA 2 and BA 3, each containing one piperidine moiety, which have shown high selective cytotoxicity against the above-mentioned cancer cell lines with average ICCF-7/ADR . Complemtitution . Collect50 = 5 µM) compared with BA, while the tri- and tetra-rhamnose derivatives expressed no sign of cytotoxicity in tested cells [50 > 50 µM), a reaction intermediate, the C28-benzyl ester of the BA-diosgenin bioconjugate (BA 12), showed promising effects against the CEM cell line, with an IC50 = 6.5 µM [50 of 4.25 µM after photostimulation in the presence of glutathione [The general approach to optimize BA bioavailability through chemical derivatization is to graft hydrophilic groups to the core structure in order to increase its hydrophilicity. Thereby, Sylla et al. obtained a series of C3-O-rhamnose-BA conjugates and tested their in vitro effects against DLD-1  and WS1 (skin fibroblast) cell lines. Among the evaluated bioconjugates, compound BA 10, 3-O-α-L-rhamnopyranosyl-(1->4)-α-L-rhamnopyranosyl-BA, exerted selective and superior cytotoxicity against colorectal cancer cells  was found to be the most active in vitro and was further associated with miltefosine, an antileishmanial drug, in order to identify a potential synergistic activity; unfortunately, the compound induced a slight decrease in the ICne alone . When anne alone . Among tne alone , who tes50 = 0.89–7.44 µM), as well as lower cytotoxicity against the tested non-cancer cells (IC50 = 17.76–27.21 µM) [50 values within the micromolar range , HepG-2 (hepatocarcinoma), BGC-823 (gastric cancer), and SY-SY5Y (neuroblastoma) cell lines. The results revealed the following aspects regarding the SAR in the series: (1) the esterification of C28 with benzyl alcohol is favorable only when the C3 position simultaneously contains a piperazine heterocycle (BA 22)—otherwise, it leads to inactive derivatives; (2) the oxidation of C3 into a keto group, combined with the introduction of a cyclohexyl-amino-ethyl, cyclopentyl-amino-ethyl, or 1-ethyl-piperidine moiety (BA 23–25), at the C28 position is beneficial for a cytotoxic effect against HeLa and SK-SY5Y cell lines . A BA-C27.21 µM) . Therefoar range  and reve50 values ranging between 0.81 and 2.62 µM [In a similar manner to the previously presented betulin, the triazole moiety was exploited for the derivatization of BA; a series of N1-substituted-1,2,3-triazole compounds were used as esterification partners for the C28-carboxyle group, resulting in derivatives with potential anticancer effects. In vitro tests against 4T1 (breast cancer) and MIA-PaCa-2 (pancreatic cancer) cell lines allowed for a SAR analysis, which revealed that the free C3-OH is essential for the cytotoxic activity since its oxidation into the keto group or esterification with succinic acid significantly reduced such effects. Furthermore, it was also noticed that some derivatives containing either pyrazinyl or indolyl moieties fused at the C2-C3 positions did not produce a significant effect on tested cells. Collectively, these findings enabled the selection of four lead molecules, each bearing a free C3-OH group and an N1-substituted-1,2,3-triazole moiety at the C28 position (BA 31–34), all exhibiting IC 2.62 µM . In the  2.62 µM , thus su50 values below 3.0 µM against MCF-7 (breast cancer) and TET21N (neuroblastoma) cell lines. The acetylation of C3 alone resulted in one BA derivative with low in vitro cytotoxicity, thus highlighting the key role played by the cationic group in exercising the cytotoxic effect [50 = 2.40 µM) compared with the parent BA, the authors reported the preparation of a semisynthetic compound with low selectivity toward cancer cells [50 ranging from 3 to 7.5 µM. The active conjugate differed from the other compounds by being substituted with two hexyl fragments of the safirinium P moiety, which is linked through a piperazinyl ring at the C28 in BA scaffold, a substitution that could favor the transmembrane passage compared with substitution with ethyl or dodecyl moieties. Interestingly, while it is well known that BA possesses mitochondria-targeted effects and substitution with cationic moieties usually boosts such effects, the study revealed that the safirinium core could not penetrate the cell membrane; therefore, its BA-conjugate accumulated inside the endoplasmic reticulum, which seems to mediate its cytotoxicity [The synthesis of new derivatives using butanoic or triethylene glycol as spacers to attach a cationic triphenylphosphonium group at the C28 position of BA was conducted by Nedopekina et al., who introduced the acetylation of the C3 position in some derivatives, thus achieving cytotoxic derivatives (BA 36–40) with ICc effect . Likewisc effect ,72 of BAc effect . The trier cells . The cattoxicity . Collect50 = 5.9–8.5 µM) against either KB or HepG2 cells compared with the parent compounds. Of note, two of these compounds were also simultaneously subjected to C3 structural modifications: esterification with succinic acid (BA 48) and oxidation into the keto group (BA 50), thus suggesting that these alterations could add to overall cytotoxicity against tested cells [Plasmodium falciparum-infected erythrocytes and human cytomegalovirus (HCMV) hosted inside human fibroblasts. The study showed that one derivative obtained through the esterification of the C3 position of BA with artesunic acid (BA 51) exhibits a potent antimalarial and antiviral activity. Although the derivative showed stronger antimalarial effects compared with BA, the compound was less potent compared with artesunic acid and chloroquine phosphate, the latter being used as a standard drug for the antimalarial effect. Moreover, the antiviral effect of the same derivative was superior to both BA and the standard antiviral drug ganciclovir [One of the most frequent approaches in drug design consists of the synthesis of bioconjugates containing two bioactive components in order to reach an overall increased therapeutic effect, based on the combination of two individual mechanisms of action. In this line of investigation, Bori et al. synthesized an ester bioconjugate (BA 46) of bevirimate with azidothymidine, a nucleoside reverse transcriptase inhibitor that is linked through a 1,2,3-triazole moiety in the C28 carboxyl group of bevirimate; the bioconjugate revealed superior anti-HIV properties compared with the semisynthetic triterpenoid . Within ed cells . Furthered cells . This hyciclovir .50 = 1.30–2.24 µM) compared with BA, thus suggesting that C3 acetylation combined with C28-grafted linear alkyl amide could enhance the transmembrane passage and, consequently, the activity of the bioconjugate [Previous studies have found the combination of BA and cisplatin to exert a synergic cytotoxic effect against several cancer cell lines ,80. In oonjugate . Continuonjugate . These i50 = 5.20 µM) [50 = 4.70 µM and OA-CD 2: IC50 = 6.50 µM) differ only by the presence of a methyl or hydroxyl substituent on the triterpenic scaffold, showed the most promising antiviral effects without associated toxicity against host cells, thus suggesting the occurrence of high antiviral selectivity that should be further investigated [Given the assumption that the poor BA oral bioavailability is caused by its low water solubility, BA was conjugated with macromolecular compounds, such as cyclodextrins (CD), in order to achieve higher hydrophilicity. Although cyclodextrins are frequently used to incorporate lipophilic molecules, such as triterpenic acids, inside their inner cavity, the outer surface could also be used to obtain numerous derivatives with enhanced biological activity . One of 5.20 µM) . Continu5.20 µM) . The aspstigated .Although the literature on the topic highlights many BA derivatives with superior biological activity, studies that have obtained derivatives with diminished activity compared with either BA or standard cytotoxic drugs should also be brought to attention in order to limit the waste of resources for fellow researchers. For example, Khlebnikova et al. obtained C28-modified F-containing-2-acylcycloalkane-1,3-diones-BA conjugates with lower cytotoxicity against MCF-7 (breast cancer) and U-87 MG (glioma) cells compared with doxorubicin . Similar50 = 3.3 µM), K562 (IC50 = 3.6 µM), and K562-TAX (IC50 =3.9 µM) [Staphyloccocus aureus compared with the parent compound, thus indicating that aromatic or liphatic N-containing heterocycles could be used for the synthesis of antimicrobial BA derivatives [Given the reported key pharmacophore role played by the triazole moiety, it was employed by Sidova et al. in the synthesis of BA-4-substituted-1,2,3-triazole derivatives via Cu-catalyzed Huisgen 1,3-cycloaddition to the C30 allylic group using C3 acetylated BA-derivatives as starting compounds; when tested in vitro against a panel of eight cancer and two normal cell lines, compound BA 53, containing an acetylated C3 and a 4-(2-formylphenyl)-1,2,3-triazole in the allylic position, showed significant cytotoxicity as well as selectivity against several types of leukemia cells such as CCRF-CEM (IC=3.9 µM) . The insivatives .Another previously reported promising approach was the insertion of sugar moieties on triterpenoid scaffolds; therefore, among a series of C30-N-acetyl-D-galactosamine-triterpenes conjugates, two BA derivatives (BA 56–57) were selected and investigated in terms of cytotoxicity, revealing, however, inferior properties compared with BA and doxorubicin; despite this apparent failure in research, the two compounds showed an improved affinity for the asialoglycoprotein receptor (ASGPR-receptor), expressed mainly in mammalian hepatocytes, which is currently the target for the development of hepatotropic drugs . FurtherIn an effort to identify new pharmacophore groups for the chemical manipulation of BA, a series of biotinylated BA bioconjugates were synthesized through derivatizations in C3, C28, or C30; the bioconjugates revealed the same level of cytotoxicity as the parent compounds against CCRF-CEM (leukemia) and HCT116  cell lines ; howeverBA conjugates can also be obtained by fusing various heterocycles to the C2-C3 bond in ring A of the triterpene scaffold; among such conjugates, Xu et al. identified one derivative containing a pyrazole fused to the A-ring of BA, SH479, which showed remarkable activity regarding osteoclast differentiation compared with BA and, therefore, could be used in the treatment of osteoporosis . In addiBetulonic acid -en-28-oic acid; The introduction of piperidine or pyrrolidine nitroxyl to the C28 position of the BoA molecule through the formation of amidic bonds may result in the occurrence of optimized hepatoprotective properties compared with the parent compound. Sorokina et al. reported the synthesis of a piperidine–nitroxide derivative BoA 1;  and a pyAnother important heterocycle found in BoA derivatives with anti-inflammatory properties is the previously mentioned triazole. Vasilevsky et al. synthesized several BoA derivatives with anti-inflammatory activity, containing a substituted 1,2,3-triazole moiety at the C28 position; the activity has been attributed both to the triterpenoid scaffold and the sidechain containing the triazole moiety. The SAR evaluation revealed that the anti-inflammatory effect increases upon replacing the hexyl substituent of the triazole moiety with an aryl substituent, in particular, an acetylphenyl group, which appears in the structure of the lead compound, BoA 6 . Similar50 = 0.17 μM, an effect that has been associated with its decrease in lipophilicity [50 = 2.4 μM [The triazole heterocycle was also highlighted as a key pharmacophore in the structure of various BoA derivatives with anticancer properties. A series of derivatives of 3-acetylbetulin and betulone containing a 1,2,3-triazole moiety were synthesized through a “click” reaction in the C28-COOH position of triterpenic compounds and assessed in terms of cytotoxicity against three cancer cell lines, C-32 (melanoma), T47D (breast cancer), and SNB-19 (glioblastoma). The study reported that, as a general rule, the substitution of the triazole moiety with carbonyl or acyl substituents such as acetyl induces stronger anticancer activity against the C-32 amelanotic melanoma cell line compared with substitution with aryl substituents. Moreover, a BoA conjugate with 3′-deoxythymidine-5′-yl (BoA 9) proved to be the lead compound against the SNB-19 (glioblastoma) cell line, with an IChilicity . In addi= 2.4 μM . Another= 2.4 μM . Unfortu= 2.4 μM . A series of indole- and pyrazine-fused ring A derivatives of lupane, oleanane, ursane, and dammarane-type triterpenoids were synthesized from their corresponding 3-oxo-derivatives; the C2–3 indole triterpenoid derivatives were obtained by Fischer indolization of 3-oxo phytocompounds with phenylhydrazine, while the C2–3 pyrazine derivatives were obtained by reacting the 3-oxo compounds with ethylenediamine. The newly synthesized hybrids were tested for their inhibitory activity against α-glucosidase, allowing some conclusions to be drawn in terms of SAR; thus, a SAR analysis revealed that the main triterpenic core significantly impacts the inhibitory activity, as indicated by the higher efficacy of lupane, oleanane, and ursane indole derivatives compared with their allobetulin and dammarane indole analogs. In addition, the activity of 2,3-indolotriterpenoids exceeded that of their corresponding 3-oxo-derivatives; particularly, 2-3 indolobetulonic acid (BoA 14) exhibited strong α-glucosidase inhibitory effects compared with BoA, proving to be 221-fold more active than acarbose .Ursolic acid (UA) is an ursane-type triterpenoid found in nature either in its free form or as the aglycone of triterpenoid saponins; it can be extracted from a large a variety of plants, but especially from apple juice; moreover, it was reported to exhibit a wide spectrum of pharmacological activities, including antitumor, antioxidant, anti-inflammatory, cardioprotective, antiviral, etc., effects . Taking 50 values ranged between 9.8 and 16 μM, indicating that the anti-inflammatory effect of each compound depends on the number of sugar units attached to the main skeleton [The rhamnose moiety, previously reported as part of the pharmacophore group due to the cytotoxic activity of other triterpenes, was also used for the derivatization of ursolic acid; the resulting saponins were subjected to in vitro tests against human colorectal adenocarcinoma (DLD-1) cells and healthy human skin fibroblasts, while their anti-inflammatory activity was quantified by assessing the NO production. The experimental data showed that the presence of a single or di-rhamnose unit did not improve the overall cytotoxic activity of the molecule compared with ursolic acid; moreover, the presence of three- and tetra-rhamnose units completely abolished the compounds’ cytotoxicity. In contrast, all derivatives, UA 1–4 , effectiskeleton . 50 = 22.9 µM) and UA 6 (IC50 = 1.55 µM) containing one furoxan ring linked to the triazole moiety. In addition, UA 6, obtained from UA 5 by acetylating the C-3 hydroxyl function, showed higher selectivity (S.I. = 6.72) for MCF-7 cells compared with both ursolic acid and doxorubicin. However, their biological activity was less effective against U-87-MG (multiforme glioblastoma), HepG2 (liver carcinoma), and A549 (lung carcinoma) cells compared with the parent compound. Moreover, the SAR analysis revealed that the introduction of an additional ester spacer between the triazole group and the main skeleton led to a significant decrease in biological activity. Betulonic acid derivatives with furoxan functions linked to the main skeleton through an amino acid linker showed no cytotoxic properties against the tested cancer cell lines. However, 1,2,3-triazole and 1,2,5-oxadiazole betulone hybrids showed selective cytotoxic activity against MCF-7 cell lines compared with the parent compound, demonstrating the importance of the triazole and oxadiazole functions toward the biological activity of the compounds [The relevance of nitrogen-containing heterocycles regarding the cytotoxic effects of semisynthetic triterpenes was confirmed for ursane and lupane derivatives as well. 1,3,4, and 1,2,5-oxadiazole and furoxane moieties were attached to the triterpenoid scaffold through plain or modified triazole linkers, which form esteric bonds with the C3 or C28 carboxyle groups. Some derivatives contained a combination of triazole and oxadiazole functions attached to the ursane and lupane skeletons via a succinate linker. The furoxane derivatives presented an additional ester linkage bond that connected the triazole linker and the furoxane fragment to the C-28 position of the triterpenoid frame. In vitro tests against several cancer cell lines revealed the strong cytotoxic activity in MCF7 cells (breast carcinoma) of compounds UA 5 , MCF-7 (breast cancer), HT-29 , and A2780 (ovarian adenocarcinoma). The results revealed that the parent triterpenoids showed little to no cytotoxicity against any cell line. However, the most effective cytotoxic effects against all cancer cell lines were proven for all 3-O-acetylated piperazinyl derivates amidic derivatives. The BODIPY hybrids exhibited little cytotoxicity; only the 3-O-acetyl-betulinic acid-derived BODIPY FL conjugate of UA (UA 7) exhibited a significant cytotoxic effect against the MCF-7 cell line with an EC hybrids .Lagerstroemia speciosa L. (banaba) with significant pharmacological potential in treating diabetes due to its insulin-like properties, which made it known as phyto-insulin [Maslinic acid (MA) is a pentacyclic triterpene occurring in several vegetables and fruits that are part of the Mediterranean diet, exerting a wide spectrum of pharmacological activities such as antitumor, antidiabetic, cardioprotective, neuroprotective, antiparasitic, and growth-stimulating effects . Corosol-insulin ; in addi-insulin . The mos50 = 499.06 μM) compared with acarbose (IC50 = 606 μM), thus revealing the importance of the free hydroxyl function in the piperazine ring regarding the hypoglycemic activity. This behavior led the authors to the conclusion that the presence of a free hydroxyl or amino group in the piperazine moiety attached to the main skeleton significantly enhances the α-glucosidase inhibition. In a series of 1-deoxynojirimycin derivatives, it was noticed that CA conjugates exerted higher α-glucosidase inhibitory activity compared with MA derivatives, while both were more effective than the parent compounds. Moreover, a SAR assessment revealed that the length of the carbon chain linker plays a crucial role in the hypoglycemic activity of the compounds; hence, a longer hydrophobic carbon chain causes a decrease in the inhibitory activity [One of the biggest challenges in managing type 2 diabetes is to control the amount of carbohydrate intake; inhibitors of α-glucosidase might ameliorate hyperglycemic episodes by inhibiting the conversion of oligosaccharides into monosaccharides, which, in turn, trigger high blood sugar. Unfortunately, conventional α-glucosidase inhibitors fail to provide high patient compliance due to several side effects, thus creating the need to develop more natural alternatives to modulate blood sugar levels . Such naactivity .Oleanolic acid  is an oleanane-type pentacyclic triterpenoid that can be found in nature as a free acid or as the aglycone of triterpenoid saponins . The phyReferring only to the period of time covered by the present review, one must cite the study of Medvedeva et al., who synthesized lupane- and oleanane-type triterpenoids by conjugating azidothymidine fragments to the main skeleton at C-17 and C-20 through 1,2,3- triazole linkers; when tested against several cancer cell lines, including HCT-116 colon, A498 kidney, PC prostate, HL-60 leukemia, RPMI-8226 myeloma, and SR lymphoma cancer cells, the results revealed that most compounds exhibited high cytotoxic activity against all types of cancer cells. Moreover, OA 1 , C-28 (hydroxyl/carboxyl group), and C-30  or the extension of the main scaffold by fusing various heterocycles with the A-ring of the phytocompound. Many such derivatives also contain linker moieties that connect the triterpenic scaffold with their reaction partners; one such linker, triazole, stood out as a key pharmacophore for the overall biological effect.Considering the high number of studies that aim to achieve the feasible synthesis of compounds of natural origin with significantly improved pharmacological activities, one may hope to discover suitable alternatives to current treatments. However, there are still important issues that need to be addressed. Firstly, more elaborate in vivo studies have to be designed in order to assess the safety and efficacy of the newly synthesized conjugates; secondly, the underlying mechanism of action for both the semisynthetic derivatives, as well as their natural parent compounds, has yet to be fully elucidated. Despite these challenges, triterpenoid conjugates may represent promising candidates for future clinical trials.


Commercial multicolour fixed immunophenotyping panels can improve flow cytometric diagnostic immunophenotyping repertoire.This study validated the commercially available, standardised Beckman Coulter lyophilised DURAClone RE panels to discriminate specific haematolymphoid subtypes.n = 11) or normal bone marrow haematogones (n = 9); CLB panels established haematolymphoid subtypes of mature B-cell lymphoproliferative disorders (B-LPD) (n = 20), while PC panels detected plasma cell dyscrasias (PCD) (n = 17). Flow cytometer setup and data interpretation to discriminate normal and aberrant immunophenotypes were per manufacturer’s instructions.We compared the diagnostic capability of the DURAClone acute leukaemia B (ALB), chronic leukaemia B (CLB), and plasma cells (PC) panels to the predicate second-line panels in Charlotte Maxeke Johannesburg Academic Hospital, Johannesburg, South Africa, from April to August 2020. Clinical diagnostic concordance between the in-house second-line immunophenotyping (the predicate method) and DURAClone was established. The ALB panels tested for precursor B-cell acute lymphoblastic leukaemia , mature B-LPD  and PCD.The DURAClone CLB exceeded the predicate second-line performance, offering extended second-line diagnostic discernment of mature B-LPD subtypes and discernment of CD5+ B-LPD from other non-CD5+ (or CD5–) B-LPD; likewise, the PC panels enabled discovery of PCD. While ALB testing offered no additional diagnostic advantage over existing predicate investigation, CD58 did offer additional information to discern haematogones from B-ALL. Multicolour fluorochrome-conjugated antibody panels generally require antibody concentrations and fluorescence intensity optimisation through titration techniques.5 These assays can be technically challenging and error-prone due to the addition of multiple individual liquid reagents requiring manual pipetting,6 thus requiring laboratory technicians skilled in titration technique and flow cytometry,7 including fluorescence-minus-one, colour compensation, and other technical flow cytometry issues.Flow cytometry immunophenotypic analysis is a powerful diagnostic and research tool for investigating haemato-lymphoid malignancies, such as leukaemia, lymphoma, and plasma cell dyscrasias (PCD). Recently, multicolour flow cytometric immunophenotyping has become invaluable for improving detection and detailed immunophenotyping during diagnosis and detection of low frequencies of abnormal or aberrant cell populations during minimal residual disease assessment.13 like the Beckman Coulter (BC) DURAClone inventory, including the identification of normal and abnormal B progenitor cells (RE ALB),14 various chronic B-cell lymphoproliferative disorders including CD5+ chronic lymphocytic leukaemia (RE CLB)15 and normal or abnormal plasma cells (RE PC) panels, offer an attractive alternative to the locally developed, in-house multicolour panels described. Commercial multicolour panels also usually offer the advantage of a standardised instrument and analysis protocols; preset software-guided controls are available to establish optimal standardised instrument settings, verified with abnormal and normal stabilised blood material controls.9 In addition, these commercial panels are typically fixed panels supplied in a stable, dry (lyophilised) format with predetermined antibody combinations in a single tube9; these qualities enhance laboratory efficiency, reduce technical laboratory error and simplify reagent inventory. Some commercial panels also offer some flexibility in marker combinations by providing ‘open’ fluorescence channels15 so that liquid drop-in markers can be added to the ‘fixed’ panels to accommodate unique and specific diagnostic requirements of the investigating laboratory.Commercially available, standardised, lyophilised, fixed and pre-titrated antibody panels,16 multicolour panel system . Although this method provides excellent coverage to discover most acute leukaemias,16 the ClearLLab 10C system lacks additional markers to further identify and discern specific haematolymphoid subtypes, especially within the group of mature B-cell lymphoproliferative disorders and PCD. Specifically, although the BC ClearLLab 10C can easily discern normal B-cell precursors or haematogones from similar immunophenotypes present in minimal residual B-cell acute lymphoblastic leukaemia (B-ALL) disease, the CD58 marker is not included. However, it may be helpful to discriminate disease from normal B-cell precursors during minimal B-ALL disease assessment.2 The ClearLLab 10C system also lacks markers to discern CD5-positive and CD5-negative mature B-cell lymphoproliferative disorders (LPD), using markers such as CD23, CD43, CD79b, CD81, and ROR-1 recommended for classification by Rawstron et al.18 Investigation of PCD is also limited if investigated only with the ClearLLab 10C system which lacks CD138, an absolute requirement for identifying plasma cells together with CD3819. In our laboratory, we have developed several 2–4-colour in-house, second-line immunophenotyping panels to identify the specific haematolymphoid subtypes mentioned, including markers such as CD23 and FMC-7 for mature B-cell LPD and CD138 with CD56 and CD200, to diagnose PCD. These, however, require a separate repertoire of liquid reagents and necessary flow cytometry technical expertise to set up and run the tests. For a busy site, such as our unit, which processes over 400 samples per month, standardised commercially available panels that can supplement second-line immunophenotypic investigation after ClearLLab10C investigation could dramatically increase the repertoire of markers tested and also improve workflow efficiency in our site.Our laboratory previously established the positive impact on quality, laboratory workflow and diagnostic usefulness of the commercially available, fixed and standardised BC ClearLLab 10CExisting predicate second-line investigation utilises locally established, in-house, non-standardised and manually assembled liquid fluorochrome-conjugated antibody panels. The primary aim of this study was to evaluate the fixed and standardised, pre-titrated commercially available DURAClone RE ALB, RE CLB and RE PC panels  as a comprehensive and compact alternative to our existing second-line immunophenotypic investigation of haematological neoplasms. There were two parts to this study. First, we compared the DURAClone CLB and PC tubes to the laboratory’s in-house predicate 2–4-colour method for the following markers: combinations of CD10, CD23, FMC7 and CD22 to discern mature B-cell lymphoproliferative disorders, or CD19, CD38, CD56, CD200 and CD138 to characterise plasma cells. Secondly, we verified manufacturer-described expression for those additional markers that were not included in the older predicate 2–4-colour method, such as verifying the expression of CD43, CD79b, CD81 and ROR-1 in the CLB tube and CD38 and CD138 with CD27, CD81 and CD56 included in the DURAClone PC tube against expected expression in normal leucocyte population counterparts. We also validated the overall diagnostic outcome of the ALB tube; here, we asked whether the CD58 included in the ALB tubes offered any additional diagnostic advantage in identifying a B-ALL that had been identified in the first-line ClearLLab 10C investigation.The University of the Witwatersrand Health Research Ethics Committee approved the study (Ethics Clearance number M1704129). The study’s objective was to validate the commercially available Beckman Coulter DURAClone PC, CLB and PC fixed-tube, pre-titrated panels as an alternative second-line diagnostic workup to our existing in-house second-line immunophenotypic investigation. The interpretation of flow cytometric data and consequent clinical diagnostic outcomes of the existing in-house, second-line, non-standardised panels were compared to the overall clinical diagnostic outcomes of the DURAClone analyses.20 The work was undertaken from April 2020 to August 2020 and conducted in the National Health Laboratory Service flow cytometry laboratory at the Charlotte Maxeke Johannesburg Academic Hospital in Johannesburg, South Africa. This laboratory performs routine immunophenotypic investigation of leukaemias and lymphomas for both children and adults, referred from Charlotte Maxeke Johannesburg Academic Hospital and other hospitals within the University of the Witwatersrand service precinct, as well as from other centres around South Africa. Morphological assessment of peripheral blood, bone marrow and trephine samples (from the same patients whose samples are tested in the flow cytometry unit) is performed in the sister haematology laboratory; auxiliary molecular and cytogenetic investigations are also performed on site. The laboratory participates in the United Kingdom National External Quality Assurance Scheme  Leukaemia Immunophenotyping Part 1 and Part 2 (interpretation) proficiency testing scheme.21This study was a prospective observational cohort study conducted according to the Standards for reporting Diagnostics Accuracy.17 or PCD22 requires second-line testing using markers that are not included in the first-line testing. For this study, in-house second-line testing specifically assessed CD23 and FMC-7 expression on mature B-cells or CD38 and CD138 expression on plasma cells. Specifically, in the context of B-LPD and in comparison to our in-house second-line investigation that included CD23 and FMC-7, could DURAClone CLB testing with ROR-1, CD20, CD43, CD79b, and 81 replace CD23 and FMC-7 in discerning CD5 B-cell chronic lymphocytic leukaemia (CLL) from other CD5 and non-CD5 expressing B-LPD.18Immunophenotypic testing of all samples at the Charlotte Maxeke Johannesburg Academic Hospital flow cytometry unit is two-tiered. Firstly, an initial first-line workup is performed with BC ClearLLab 10-C . The first workup identifies most acute myeloid and lymphoid leukaemia subtypes, distinguishes early and mature T-cell or B-cell lymphoproliferative disorders, and hints at the presence of a PCD (if a population of brightly expressing CD38 cells is noted). A limitation of this ClearLLab 10C system is that further immunophenotypic characterisation of mature B-cell lymphoproliferative disordersn = 13), bone marrow (n = 43), and a pleural fluid sample (n = 1) collected from paediatric and adult patients. For DURAClone ALB testing, 20 bone marrow aspirate samples were tested. Eleven of these patients had a diagnosis of B-ALL (n = 11), while nine had immunophenotypically normal haematogones. One of the nine was a follow-up of a B-ALL with normal haematogones and no evidence of residual disease. Twenty samples diagnosed with a B-cell lymphoproliferative disorder by predicate ClearLLab 10C and in-house second-line were referred for DURAClone CLB testing. The CLB testing set comprised 12 bone marrow aspirate samples, seven peripheral blood samples, and a single pleural fluid sample. Due to the decommissioning of the older FACSCalibur flow cytometer during the period of study (the instrument used to undertake the predicate in-house second-line testing), only 17 samples with a diagnosis of a PCD by ClearLLab 10-C and in-house second-line investigation were tested by DURAClone PC panels.After all routine testing with ClearLLab 10C and in-house second-line testing , samplesTM  and the FACSCalibur flow cytometer  were used during this study. The NaviosTM flow cytometer was used to analyse the predicate ClearLLab 10C as well as the test DURAClone panels. Internal quality control on the NaviosTM included daily assessment of background contamination, cellular events carryover between tubes, and fluorospheres acquisition to verify the flow cytometer optical alignment and fluidics . In addition, ClearLLab™ Normal and Abnormal process control cells  were used to verify sample processing, acquisition, and analysis. ClearLLab 10C panel acquisition setup was achieved by applying target values set in the manufacturer manual and using BC FlowSet Pro beads to adjust the voltages that enabled optimal detection and separation of dim and bright antigens. After pilot testing (data not shown), the NaviosTM instrument ClearLLab 10C settings were deemed appropriate for the DURAClone multicolour data acquisition and analysis.The NaviosTM process control  using four monoclonal antibodies, CD14 FITC, CD13 PE, CD45 PERCP and CD3 APC , was done.The FACSCalibur flow cytometer was used to acquire the predicate in-house second-line 2–4-colour fluorescence panels using CellQuest software . Quality control performed on the BD FACSCalibur included daily assessment of background contamination, carryover, acquisition, and analysis of manufacturer-recommended 3-colour and APC Calibrite beads . Further acquisition and analysis of Immunotrol3 and 0.0372% ethylenediaminetetraacetic acid; Merck, Darmstadt, Germany) in a 15 mL conical centrifuge tube for 15 min at room temperature. After incubation, samples were spun at 3000 g for 3 min, the supernatant was decanted, while the pellet was washed four times with 14 mL phosphate-buffered saline at pH 7.3 ± 2  containing 0.09% sodium azide (NaN3)  and 0.2% bovine serum albumin . Following washing, samples were reconstituted to 0.5 mL with phosphate-buffered saline, and the white cell count was determined. Cells were then diluted (or concentrated) to achieve the recommended cellular concentration of ≤ 10 000 cells/µL for monoclonal antibody incubation. Subsequently, 100 µL of this cell concentrate, which contained approximately 106 cells, was added to the ClearLLab 10C and in-house panels  with 14.5 mL isotonic ammonium chloride pH 7.1–7.4  events were collected. Paint-A-Gate software  was used for analysis of raw FACSCalibur flow cytometric data with a primary gating focus using CD45 and side scatter.23 First, doublets, debris and unlysed red blood cells were excluded, and, where possible, a neoplastic target population was identified in a primary gate using CD45 and side scatter. Thereafter, secondary gating focused on identifying the same target immunophenotype across each of the T, M1 and M2 panels.16 Finally, simultaneous identification of normal populations in the background was based on local in-house developed gating strategies to identify granulocytes, monocytes and mature lymphocytes using CD45, side scatter characteristics and specific regular expression of the markers studied  events were acquired on the Navios. Kaluza C™ version 1.1  software was used to analyse all raw listmode data to facilitate clinical interpretation. Kaluza C™ ClearLLab B, T, M1 and M2, and DURAClone panel analysis protocols were developed according to manufacturer specifications. studied .23 The relative intensity of expression of cell surface antigens was used to discriminate between normal and aberrant immunophenotypes according to manufacturer specifications. Positivity (or no expression) of individual markers was established, and overall haematolymphoid immunophenotypes sub-classified according to the established positive markers outlined in the World Health Organization (WHO) 2016 classification of tumours of haemopoietic and lymphoid tissues.24 Specifically, expression of CD27, CD43, CD56, CD58, and CD81, which were not included in the predicate ClearLLab 10C and in-house second-line panel analysis, were verified on background  populations as evidence of a satisfactory internal positive control and proof that the reagents met the manufacturer’s performance specifications. In the ALB analyses, typical secondary backbone markers, including co-expression of CD19/CD10/CD34 enabled further characterisation of markers CD20, CD38, and CD58 and established the presence of normal precursor B-cells (haematogones) or abnormal precursor B-cells. Likewise, for the DURAClone RE CLB, dual expression of either CD19/CD5 or CD19/CD20 was used to identify the ‘target’ or neoplastic B-cell population before specific characterisation of CD43, CD79b, CD81 and ROR1 expression. Plasma cells were defined in the DURAClone RE PC panel by the dual expression of both CD38 and CD138; a normal or malignant plasma cell immunophenotype was subsequently noted following interrogation of markers including CD81, CD27, CD19, CD200, CD56, and CD45.A similar approach was used for each of the comparative second-line DURAClone RE ALB, RE CLB, and RE PC listmode data analysis but additionally incorporated manufacturer-recommended gating strategies for clinical interpretation of data.Clinical diagnostic outcomes were collated into Microsoft Excel  spreadsheets. Marker expressions and specific clinical diagnoses noted for the DURAClone RE ALB, RE CLB, and RE PC panels were compared to the clinical diagnostic outcomes from the existing in-house 2–4-colour antibody panels described . The agrTwenty samples were tested with the DURAClone ALB panel. The DURAClone RE ALB evaluation on both routine diagnostic precursor B-ALL samples, and patient samples who were being followed up after therapy for precursor B-ALL, revealed 100% positive and negative agreement to ClearLLab 10C reported outcomes . The estn = 20). All CLL cases (n = 16/20) showed under-expression (weak) of CD81, CD79b and CD20 (n = 1) showed a bright expression of CD79b. Three cases (15%) evaluated could not be definitively sub-classified with the in-house second-line panel analysis or the DURAClone RE CLB reagents. An additional control sample  (n = 1) PCD had positive surface CD117 expression  . A singlpression  on the a16 fixed-tube, standardised, pre-titrated reagent system, ClearLLab 10C , and ClearLLab LS11 were recently implemented to replace our outdated FACSCalibur/Paint-a-gate method for routine immunophenotypic investigation of haematological neoplasms at our laboratory. This new system provides flow cytometric workup for haematological neoplasms and enables processing up to 400 samples a month. Previously, leukaemia flow cytometry testing was limited to simple 2–4-colour analysis; sample setup was manually intensive and error-prone. Systems previously implemented into our laboratory streamlined standardised and automated testing and improved workflow and quality of reporting.27 Similarly, implementing the ClearLLab 10C and LS system in our laboratory16 has markedly improved workflow and minimised errors while providing standardised and substantially better quality-controlled sample and data acquisition and comprehensive data. The latter allows for improved target population identification, especially for small populations.The recently evaluated,28 and could theoretically be assembled as required in the lab to provide additional marker panels needed to complete full patient immunophenotypic investigation. However, setting up quality-controlled multicolour panels in low- and middle-income countries is both challenging and less reliable16. The ClearLLab system facilitates detailed diagnosis across a broad range of acute leukaemias, and is sufficiently sensitive to detect the presence of a mature B-cell LPD, but it lacks specificity to discern and diagnose different mature B-cell LPD9 or confirm PCDs. For example, although the B-cell panel is useful to differentiate precursor B-cell from more mature B-cell LPD, the panel does not accommodate sufficient markers to discern different types of mature LPDs, including CD23, CD43, CD79b and FMC-7 markers necessary to distinguish B-cell LPD per the WHO classification.24 Likewise, markers, such as CD58, for diagnosis for subsequent follow-up of precursor B-ALL, or CD79a and CD81, would be of value in the B-cell panel to discern neoplasm from haematogones. Plasma cell dyscrasias are also not easily identified in the ClearLLab system, which lacks CD138 and other markers useful for diagnoses and follow-up of PCD.30 Although CD38, CD56, and CD45, used to identify plasma cells, are included in the ClearLLab panels, plasma cell identification is not definitive. Specifically, CD56, included in the T-cell panel, is assessed separately from CD38 (included in the B-cell and M2 panels). A pre-titrated-monoclonal panel including all three markers would suitably enhance the ClearLLab system; the DURAClone panels used in this study provided this supplement format.Complementary and supplementary multicolour panel options are published32 along with CD81 expression.31 CD58 in the index test ALB tube proved to be an excellent adjunct for assessing precursor B-cells and was helpful in discerning abnormal precursor B-cells from normal precursor B-cells (haematogones)31. Distinct discordant CD123 and CD34 expression patterns have also been described on haematogones and can be used in a strategy to determine B-ALL minimal residual disease.32 CD123 was not however included in the ALB tube. In our predicate method, the ClearLLab 10C system, co-expression of CD123 with CD34 and CD19 assessment within the ClearLLab M2 panel, used to identify precursor B-cells, together with CD19, CD10 and CD34 co-expression in the ClearLLab B-cell tube, therefore offers more diagnostic information than the DURAClone ALB tube alone for the follow-up of precursor B-ALL and residual disease assessment. The ClearLLab 10C system was developed to diagnose early and later B-cell lymphoproliferative disorders; these 10-colour panels are fixed. Thus, additional markers cannot be tested for in the panels. However, additional markers can be added to the RE ALB, fine-tuned toward detecting early B-cell precursors only. CD22 and CD123 could be valuable additions to the ALB panel. For example, a liquid reagent drop-in of CD22-APC in the free APC fluorescence channel of the DURAClone RE ALB panel could provide a baseline assessment of CD22 expression on B-ALL blasts, which would be useful for deciding on anti-CD22 targeted therapy in B-ALL patients. CD123 could also be added in a second available channel to enable further discrimination of disease (B-ALL) from normal haematogones  and clinical diagnostic outcomes agreement. The additional markers in the DURAClone RE CLB panel, including CD81, ROR1, CD79b and CD43 which are not included in the laboratory predicate ClearLLab system, specifically the B-cell tube, met the requirements for intended use and the manufacturer performance specifications. The DURAClone RE CLB panel proved to be an excellent fixed supplementary panel to replace our outdated second-line investigation for the workup of mature B-cell lymphoid proliferations and proved to be especially useful for discerning CD19/CD5 positive haematolymphoid disease subtypes. The surface CD81, CD79b and CD20 showed consistent under-expression in CLL, while the presence of ROR1 was also helpful in discerning CLL over mantle cell lymphoma as described in previous studies,33 especially when interpreted in conjunction with CD200 expression in the ClearLLab B-cell panel.35 In the event of a CD19/CD10 co-expressing mature B-cell lymphoid proliferation, CD43 in the DURAClone RE CLB panel was shown to be a useful cell surface marker to discriminate follicular lymphoma from a high-grade B-cell lymphoma. Similarly, CD79b within the DURAClone RE CLB panel, read together with CD38 expression in the ClearLLab B-cell tube, identifies a mature B-cell lymphoid proliferation with plasmacytoid differentiation. As for the ALB panel, free channels in the panel allow for additional flexibility to investigate related markers that are regarded as useful to discern mature B-cell haematolymphoid subtypes. A liquid reagent drop-in of CD23-ECD in the DURAClone RE CLB panel could further confirm B-cell CLL and discern B-cell CLL from other CD19/CD5 co-expressing target populations.33The expression of CD5, CD19, CD20 and CD45 in the DURAClone RE CLB reagent showed excellent cross-panel marker expression, equivalent to ClearLLab 10C B panel expression, with 100% (n = 17/17) clinical diagnostic concordance (agreement). In addition, as previously described,29 CD27 and CD8130 proved to be valuable markers to discern aberrant plasma cells over normal plasma cells. Finally, the usefulness of the RE PC panel can be further extended by adding CD117-ECD30 reagents. For one sample, we added CD117-ECD reagents to the PC tube, confirming the presence of malignant plasma cells and minimal residual disease assessment.14,30.The CD38, CD138, CD45, CD19, CD56, and CD200 expressions in the DURAClone RE PC panel showed excellent cross-panel marker expression and equivalent diagnostic outcome and FACSCalibur/PAG analysis outcomes, with 100% (23 (of the particular laboratory’s flow cytometer) can accommodate data collection from the fluorochromes used in the ClearLLab 10C or DURAClone panels.Firstly, the sample size per panel evaluated is small, and further studies are needed to confirm the outcomes reported here. Secondly, the fluorochromes used in the panels are specifically designed for use on a BC Navios instrument. Therefore, the products could be used on alternative instruments only if the respective filter setupsThis study confirms that the BC DURAClone ALB and CLB panels are suitable to provide additional second-line immunophenotypic workup of precursor B-ALL and mature B-cell lymphoproliferative disorders resepectively, at disease presentation and are suitable to supplement first-line ClearLLab 10C laboratory predicate method testing. The under-expression of CD81, CD79b, and positive CD43 and ROR1 expression, noted in the DURAClone CLB panel specifically assists in distinguishing B-cell CLL from mantle cell lymphoma and other CD5 negative B-cell LPDs.The DURAClone RE PC panel is suitable for the second-line investigation of PCD. CD27, CD56, CD81 and CD200, together with CD19, CD38 and CD138 in a single analysis, were efficient in identifying aberrant plasma cells.31 in the RE ALB tube may however be a valuable adjunct for discerning normal reactive B-cell precursors from the minimal residual disease at follow-up.Although there was diagnostic and individual marker concordance, we did not find the ALB more useful for diagnosing B-ALL over our existing ClearLLab system (utilising the B-cell and M2 tubes). The inclusion of CD58Lastly, the potential benefit of using commercialised multicolour lyophilised fixed panel preparations having stable, standardised antibody reagents reduces the risks of technical error, improves laboratory efficiency, and simplifies reagent inventory. In addition, the DURAClone ‘free fluorescence channels’ provide some additional flexibility for a laboratory to use their preferred markers to establish diseases (or not) of their choice.


This study aimed to explore the experiences of adult clients with hearing loss and their audiologists in using an online tool, the Living Well Tool (LWT), during initial audiology appointments. The LWT is designed to help clients identify when and where it is most important for them to communicate effectively and live well with hearing loss. A total of 24 adult clients with hearing loss and two audiologists participated in this study. Clients were invited to complete the LWT prior to their next audiology appointment, however, most clients chose to use the LWT in-session with their audiologist. Following the appointment, clients and audiologists participated in individual qualitative semi-structured interviews to explore their experiences of using the LWT, and the extent to which the LWT facilitated person-centred care. Qualitative analysis five key themes which reflected participants’ experiences and perceptions of using the LWT: (1) the LWT enhances audiological care; (2) the LWT supports person-centred audiological care; (3) the use of the LWT should be individualised; (4) users value comprehensiveness; and (5) users value accessibility. This study demonstrated that the LWT supported the provision of person-centred audiological care, providing a flexible, comprehensive and accessible means for audiologists to gain an understanding of their clients’ needs and preferences. However, it was also noted that the use of a tool must be individualised and accessible for all. Hearing loss occurs frequently in older adults and has numerous negative consequences, including reduced access to everyday spoken communication, social isolation, depression, poor mental health, and reduced quality of life . Due to Despite widespread acknowledgement of the importance of person-centred care in audiological rehabilitation, research suggests that hearing healthcare continues to have a biomedical focus. In a video observation study, clients and their significant others often raised psychosocial concerns related to communication difficulties during appointments, however these concerns were rarely addressed, with audiologists having a tendency to focus on providing technical information and progressing a discussion about hearing aids . AlthougeHealth can be used as a way of enhancing person-centred care in healthcare . eHealthThe adoption of technology by older adults in managing their healthcare is heavily influenced by the platform’s perceived usefulness , accessihttps://apps.idainstitute.com/apps/lw_en) (accessed on 1 July 2021). The LWT is designed to promote communication and living well with hearing loss for clients, using an active problem-solving approach to focus on relevant and important communication situations and the biopsychosocial needs of the client, rather than focusing solely on hearing aids and the hearing loss. The online LWT has been designed to empower clients to prepare for the rehabilitation process. This is achieved by encouraging them to think about their communication needs prior to their audiology appointment, with a series of photographs and open-ended questions helping them to identify when and where it is most important for them to communicate effectively and live well with hearing loss. Armed with this information, the client and their audiologist can work together to map out strategies for managing the hearing loss and communication goals in a person-centred manner to ultimately improve communication outcomes  an improvement on the way it [case history] was done before” (Client 14), even providing additional case history questions: “Sometimes you [audiologists] don’t always remember to ask all the questions even if you have a sort of template that’s being followed, so it’s [LWT] probably good” (Client 16).The LWT complements traditional information-gathering practices. Within the context of typical audiology appointments, participants noted that the LWT was useful in setting the agenda and guiding the case history: “That list of [communication strategies in LWT] gives you an idea of what options you could have available” (Client 15) and “It took the focus of the hearing aid to … this is what you can do to make it better for you, so head against a wall in a noisy restaurant, turn the music down, you know you get TV headsets for the television if you’re having trouble with Scottish accents” (Clinician 1). Some client participants noted that the LWT prompted them to identify additional communication situations to discuss with the audiologist: “I hadn’t thought about the supermarket … so it does jog the memory when it needs to” (Client 8).Participants also described how the LWT was used to frame discussions around various communication situations, communication strategies, and technological solutions for improving communication in these situations. Clients and clinicians alike noted the usefulness of the LWT in helping clients to identify potential communication strategies: “positioning [myself] to hear and situations where I needed assistance” (Client 19), while another described how the audiologist used the LWT to discuss hearing aid solutions: “she was making the point how it [hearing aid] copes in this sort of situation [shown in LWT] and therefore that it [hearing aid] would be beneficial to be in terms of being able to hear better in that sort of scenario” (Client 16). Finally, participants recognised the benefit of the LWT in keeping records for both the client and audiologist: “It leaves a permanent record [of what was discussed with audiologist] ‘cause you know if you have a meeting with someone you can talk for 15 min about the problems but have no record” (Client 3).Another participant described how the audiologist used information from the LWT to discuss communication strategies which could be used in social situations: “I probably would’ve been more informed [if I had completed the LWT prior]” (Client 1). One participant reflected “What the program [LWT] said to me was, this is like pre-empting your visit. The technician’s [audiologist] got some information about you” (Client 4), while others said “I probably I came [to the appointment] prepared to be saying ‘This is the problem I’m having’” (Client 16). Others saw how the LWT could be used to help clients identify their own hearing difficulties, and even the potential use of the LWT as a communication screener: “You could screen people using the device [LWT] rather than taking history and everything when you actually saw them … and those that have a problem need to come and those who don’t have a problem may not need to come … or may not need to come so urgently” (Client 15). Interestingly, client participants also saw the benefits of the LWT in preparing audiologists for appointments, for example: “They [audiologist] might be able to pre-empt the problem … before they see the patient … and save them [audiologist] time from asking all the questions … which would make the appointment more efficient” (Client 15).The LWT could enhance preparedness for the client and clinician. One of the key benefits outlined by participants was the potential application of the LWT in preparing clients for appointments and even facilitating the help-seeking process. Despite only a small number of clients competing the LWT prior to their first appointment, many reflected that completing the LWT pre-appointment would have been valuable to the process: “it doesn’t [take more time], because you would be doing COSI [Client Oriented Scale of Improvement] goals anyway” (Clinician 2) and in fact, “it [LWT] kept me on track … rather than 20 min trying to get to the point [of the client’s visit]” (Clinician 1). Client’s also noted that “it was really just a few minutes at the start of the appointment” (Client 18) and “we [client and audiologist] didn’t have to go through a long, tedious interview” (Client 8). Clients noted that the LWT could also be a helpful tool in improving session efficiency for certain clients: “I think it [LWT] could save a lot more time with people who were maybe were a little bit reticent” (Client 11).The LWT improves appointment efficiency. Despite clinicians expressing concern that, prior to using the LWT, it would take too much time to complete, they reflected after the study that “forced the clinician to ask questions and listen to answers” (Client 7). One client noted how using the LWT “probably merged a bit into a conversation … rather than sort of being too locked into a series of questions … ensuring the content was covered naturally” (Client 17). The clinician participants agreed that the LWT prompted them to ask additional questions about the client’s use of communication strategies: “It [LWT] actually gave you more information, like what they’re actually doing [strategies] … that’s something that I probably got a lot more information than I usually would” (Clinician 2).The LWT aids communication processes. Client participants highlighted how the LWT Theme 2: The LWT Supports Person-Centred Audiological care.Analysis of the client and clinician interviews identified several ways in which the LWT supported person-centred audiological care, including facilitating the client-clinician relationship, making the discussion of sensitive matters easier, helping clients identify the biopsychosocial impacts of their hearing disability, making clients the priority, and normalising hearing experiences to help clients identify strategies.“I think it [LWT] actually engaged the clinician and the patient, or client more closely” (Client 7) and “I think it [LWT] was creating more of a rapport … and just took away from the ‘I’m the audiologist and you’re the client” (Clinician 1). One participant even reflected that the tool enabled the relationship to begin even before the first appointment: “I felt like somebody was talking to me already … the bond that existed before you even walked in here, before you even met” (Client 5).The LWT facilitates the client-clinician relationship. Both client and clinician participants agreed that the LWT played a role in building the client-clinician relationship: “It [LWT] obviates the necessity of other people pointing out to you that you’ve got hearing problems … a computer is nicely non-personal … at least when you do this sort of thing, you have to come to terms with it yourself” (Client 13). The LWT was also thought to be helpful in sensitively raising communication difficulties that clients might feel uncomfortable about: “I can understand how somebody who was a little bit hesitant might find it really really good” (Client 11), and even help facilitate more thoughtful discussions with family members about difficult communication situations: “It [LWT] helps them [family members] understand the person with the problem … they [family members] might try to assist in certain ways and not be so critical when people didn’t pick up what they were saying” (Client 15).The LWT makes discussing sensitive matters easier. In the context of audiological rehabilitation, the LWT was described by the participants as an effective means of sensitively communicating the diagnosis of a hearing loss: “they [clients] do tend to say ‘I don’t think I have any trouble’” (Clinician 1), with one client even noting that “It’s good to see that there’s different ways of looking at it [hearing assessment] now [other than solely instrumental assessment]” (Client 20). In particular, the pictures of various communication situations prompted clients to think: “Oh yeah, I have that problem” (Client 16) and were helpful in making “the client more aware of the different ranges of hearing situations and how they are currently effective or non-effective” (Client 7). Importantly, the LWT was also effective in helping clients identify their communication strengths: “There are many situations in which there are no problems … made me aware that I’m competent in a lot of ways” (Client 20). This focus on communication strengths was noted to be a particularly important given that clients may not always “think of that [positive] perspective” (Client 15).The LWT can help clients identify the biopsychosocial impacts of their hearing disability. Both clients and clinicians found the LWT particularly helpful in prompting clients to think about the broad ranging impacts of their hearing loss, and the everyday difficulties they encounter as “I think it [LWT] was creating more of a rapport so that they knew I was focusing on what their needs were … which in turn got me to focus on ‘Ok, this is what you want solved’, as opposed to me presuming what they were there for.” (Clinician 1). The LWT was also identified as a means of putting the client in the driver’s seat: “They [the client] felt more in control of giving me the information rather than often I’m like, ‘Oh, so how do you find the phone?’ So more control, they have more control I think” (Clinician 2).Clients are the priority. The LWT was identified by participants as a method of ensuring the clinician stayed focused on the needs of the client: “I think it’s [LWT] effective in that it does relate the conditions you’re having evaluated to your real life situations” (Client 21). Further, participants described how the photographs served to reassure clients that other people also experienced communication difficulties: “By looking at the pictures and realising that there’s a lot of other people in those kinds of situations … that you’re not alone kind of thing” (Client 10).Relatability of photographs can normalise hearing experiences and help clients identify strategies. Throughout the interviews, clients discussed the relatability of the photographs used in the LWT, and the effectiveness of the photographs in identifying the everyday communication difficulties resulting from hearing loss: “The only one that remotely identified with me was the crowd scene or the party scene” (Client 12). Further, some participants noted that the photographs portrayed people of a homogenous ethnicity and age: “I think they were all Caucasian people” (Client 8).Despite these positive reports about the relatability of the photographs used in the LWT, it is important to note that not all clients could relate to the communication situations depicted in the photos: Theme 3: The use of the LWT should be Individualised.Consistent with the principles of person-centred care, there was wide acknowledgement amongst participants that the use of the LWT should be individualised according to client needs because there was variability in how the LWT was used. The results also highlighted that audiologists provided valuable guidance when using the LWT and that some users value in-person services.“most of my clients didn’t do it [LWT] at home” (Clinician 1) and “I don’t think I had any [LWTs] completed in advance” (Clinician 2). One of the audiologists gave her clients the choice of how they would like to complete it, noting that “When I make the appointment I say to them ‘I can either send it to you and you can do it online or we can do it together when you come in’” (Clinician 1). The most common method of completion of the LWT, therefore, was within the audiology appointment time. Although both clinicians indicated a preference for using the LWT at the beginning of appointments “as part of the case history” (Clinician 2), clients reflected that “it doesn’t seem to matter which part of the appointment you have it in … it wouldn’t matter if it was at the start or the end really” (Client 9).The LWT is used in Varied Ways. Although the online LWT was initially designed to be used by the client at home prior to appointments, and then discussed within the appointment in the context of the client’s hearing test results, analysis indicated that the LWT was used by clients and audiologists in varied ways. Despite most clients being sent an electronic link to the online LWT, “I don’t actually recall us sort of referring back to that [LWT] per se”. Interestingly, a number of clients noted that they saw potential in using the LWT again at a future point in time: “I know how to access it so if there’s a need for me to progress if for whatever reason, if there’s any further change to my situation, well … there is somewhere I can go to, there is somewhere I can source this information” (Client 5) and “I guess you could compare those [LWT responses] overtime” (Client 17).In regard to ongoing use of the LWT, most clients reported that following completion of the LWT, the audiologists did not commonly refer back to the LWT: “I didn’t have too many where they actually came along, which is disappointing” (Clinician 2). When family members did attend appointments where the LWT was used, one family member reported “I kept as quiet as I could possibly be [during the LWT discussion] … I didn’t say very much at all” (Family Member 4), indicating that they were not actively involved in the process. One of the clinicians did note however that she thought the LWT was helpful in provoking discussions about third-party disability. Interestingly, one of the audiologists reflected that the LWT could be used to manage dominant family members: “It would be interesting to do more of the tool with the significant other to see if it stops them from being the dominant one [and bring the focus] back to the client” (Clinician 1).Family Member Involvement was Varied. According to participant reports, family members played a limited role in the use of the LWT, most often due to low attendance of family members at audiology appointments: She [audiologist] spoke to each question” (Client 2) and “asked how I coped in the various situations” (Client 12). The level of input provided by audiologists varied between clients, with audiologists acknowledging that “some people just needed more … some guidance” and “it seems to work whichever way you do it” (Clinician 2). As such, while some clients reported that the audiologist took the lead and completed the tool on behalf of the client by asking them questions: “I didn’t enter anything” (Client 8),“I just gave answers [to the audiologist’s questions]” (Client 13), others reported that “she [audiologist] turned the screen around and gave the mouse to me so I was actually writing it … I pretty much went through it [LWT] on my own … I think she was more there as a support if I needed it” (Client 20). When guidance was provided, this most often included an explanation of various communication strategies such as asking people for clarification, and at the end of appointments linking the results of the hearing test to the LWT responses: “She [audiologist] did make connections between the situations that were identified on the website and the results of the tests” (Client 21). Clients noted a preference for the audiologist to enter client responses into the LWT: “It was probably easier if she [audiologist] uses it. She could put the information in faster than someone who is at home” (Client 15), however a number of clients also noted that while they were completing the LWT with the audiologist, they could not see the computer screen (Client 13). Overall, however, the majority of clients saw value in going through the tool with the audiologist: “it would be a bit helpful if I wanted to talk through issues with someone … if people are using it by themselves for a diagnosis then you might be entering difficult territory” (Client 17).Audiologists Provide Valuable Guidance when Using the LWT. Clients reported that their audiologist provided valuable guidance when using the LWT, with audiologists talking through the instructions with clients, describing the pictures, defining the communication situations, typing in client responses, and discussing these responses throughout the process: ““I’m not one of those people who likes talking to screens” (Client 12), as well as preferring to discuss the results directly with their audiologist: “It’s probably more personalised to do it [LWT] actually with the audiologist rather than do it and give it to the receptionist and then never see it again, and don’t know whether anybody’s even looked at it” (Client 8).Some LWT Users Value In-person Services. Despite the acknowledged benefits of having access to an online LWT, some clients acknowledged that they preferred to use the tool with their audiologist in an in-person appointment. Client preferences for in-person services centred around their comfort with using technology for healthcare: Theme 4: Users value Comprehensiveness.Analysis of the interviews revealed a common finding that the users of the LWT valued comprehensiveness in a tool such as the LWT, and as such, there was potential for the LWT to be further developed to enhance its application in audiological rehabilitation.“plenty of options” (Client 11), along with “a list of different perspectives” (Client 15).The LWT is Comprehensive. Overall, participants agreed that the LWT was a comprehensive tool which offered “the audiologist a bit of a better indication of what your problems are” (Client 3), especially “if you had a problem and you felt you wanted to do a few different things” (Client 5). Participants also agreed that the LWT could be more comprehensive by asking clients to identify more potential communication strategies.There is Potential for the LWT to Ask More. Participants did acknowledge however that the LWT had the potential to ask more information and to give more information to clients with hearing loss. Participants suggested that the tool could ask for more specific information to give “examples of what you can hear and what you can’t hear” (Client 3), and providing more background information about the communication scenarios “with maybe even more of a breakdown of the different scenarios rather than just the pictures … more clarification of the pictures” (Client 10).There is Potential for the LWT to Give More. Participants also noted that there was potential for the LWT to include more communication scenarios, more specific communication strategies, giving more Theme 5: Users Value Accessibility of the LWT.Participants identified the importance of being able to readily access tools such using technology such as the LWT. Participants thus emphasised the importance of having a tool which is easy to use and is aesthetically pleasing, especially given the potential challenges of using technology.“was very straight forward” (Client 8) and “pretty easy to understand” (Client 9), especially given its logical step-by-step approach. One of the clinicians noted however that “it’s not so intuitive” (Clinician 2) and may be difficult for some clients to use. This was especially true for clients with other co-occurring conditions such as vision loss, which could affect their access to the LWT, and therefore has implications for the design of the tool, including font size: “You might make the text [of the LWT] bigger for people with poor vision” (Client 15).Usability of the LWT is Valued. It was apparent from the interviews that the usability of the LWT is an important consideration in order to maximise accessibility for users. The majority of participants noted that the LWT “When you’re faced with a website that you don’t have to think about … it means it’s [the LWT] got to be good” (Client 11). Participants also reported that the “pictures were effective in picking up [communication] situations” (Client 21). Some participants did note however that “the photos were a bit busy … and quite small” (Client 17).The Aesthetics of the LWT Enhances its Accessibility. Overwhelmingly, the participants agreed that the LWT was “well presented” (Client 17) with its design and aesthetics positively enhanced it’s accessibility: “older, much older people [who] may have a little bit of difficulty with it … if you don’t understand the computer” (Client 10). Some participants even expressed a preference for the LWT to be also provided in a “printable format” (Client 2) so they could “fill it out” (Client 15) in paper form, especially given some clients reported some technical difficulties with sending the electronic copy of the completed LWT to their clinician. Other participants with limited computer experience noted however that despite this lack of technological experience, the LWT was easy to use.Technology can be Challenging. Given the online format of the LWT, participants did make reference to the inherent challenges of an online platform, especially for The present study investigated the experiences of clients with hearing loss and their audiologists in using an online audiological rehabilitation tool, the LWT, and specifically, the extent to which the tool facilitated person-centred audiological care. We aimed to explore the experiences of clients and their audiologists in using the tool and determine the extent to which the LWT facilitates the provision of person-centred care. In doing so, we explored the effectiveness of Living Well in focusing audiologists’ talk on communication in everyday life rather than just hearing aids when working with adults with hearing loss and their families. Results of this study produced two overarching ideas. The first of these was that Living Well embodied person-centred care, as indicated by themes 1–3; the second was that users value the website’s functionality, shown in themes 4 and 5.The Living Well tool embodied person-centred care as indicated by the themes LWT supports person-centred audiological care, LWT enhances audiological care, and Use of LWT should be individualised. Participants in the present study showed the Living Well tool is adaptable to individuals’ heterogeneity by describing the variability in tool operator , how the tool is used  and how family members were or could be involved. Previous research has identified the importance of taking a person-centred approach to healthcare ; this waParticipants described how the LWT facilitated communication, facilitating the relationship between clients and clinicians, and easing the discussion of sensitive matters. This description is consistent with  characteIn the present study, clients and clinicians alike commented on the ways in which the LWT integrated with existing models and activities of care without diminishing traditional practice. Both client and clinician participants reported that the LWT improved preparedness for audiological rehabilitation, and therefore promoted greater session efficiency. Importantly, participants also reported that the LWT did not add any more time to appointments. Identifying these benefits is important given clinicians report time as a barrier to implementing person-centred care ,29,30. IIn addition to enhancing the information that is received from clients, the LWT also has capacity to help clients gain additional insight; the LWT helped clients identify the biopsychosocial impact of their hearing disability. Many clients underestimate the impact of their hearing loss until rehabilitation has commenced. The graduated nature of most cases of hearing loss means many clients do not realise what they are increasingly missing out on  and the In recognising client strengths and providing individualised care, information and its presentation can be tailored to suit the user , which cAccessibility of such programs has been commented on extensively in the literature, which has found that designing for older users requires careful consideration of layout, use of blank space and proximity and choice of colours ,39, and In terms of clinical implications, the Living Well tool provides a range of options for clinicians in future. As shown in the present research, it can be administered within the clinic with the help of the clinician, or can be used at home, either alone or with a family member. Indeed, input from family members is strongly encouraged; research has shown family-centred care improves communication within families and reduces third-party disability ,44. FamiThe present study contained some limitations. Firstly, we had limited control over which participants were invited. Clients of the participating clinics were invited directly by the audiologist; accordingly, we have no knowledge of which clients were not invited or for what reasons. Secondly, many of the interviews were conducted in the clinic of the treating audiologist, which may have influenced the interview data. Despite assurances of confidentiality, clients may have been concerned their responses could be overheard by or provided to clinicians. We conducted interviews in the clinic to maximise response rates; however, future research may be able to circumvent potential influence on interview responses by conducting all interviews in a separate setting. Finally, we aimed to gain the perspectives of family members, yet only two clients brought family members to their appointments. Although low levels of family attendance in audiology are common , increasDespite these limitations, the present study was the first of its kind in examining client and clinician experiences of using this LWT, and thus has provided important insights into how much an online tool can be used to enhance audiological care. The LWT tool provided participants with a platform to discuss their hearing and communication needs with their audiologist, in a way that has promoted person-centred care via individualised, comprehensive, accessible care that is suitable for an older audience. The use of the LWT can be expanded to provide family-centred care by including the needs and perspectives of family members.