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Original article
A parallel comparison of T-cell clonality assessment between an in-house PCR assay and the BIOMED-2 assay leading to an efficient and cost-effective strategy
  1. Szu-Yin Kuo1,
  2. Hongxiang Liu2,
  3. Yung-Liang Liao1,
  4. Sheng-Tsung Chang1,
  5. Yen-Chuan Hsieh1,
  6. Betty Angela Nuako Bandoh2,
  7. Ming-Qing Du2,
  8. Shih-Sung Chuang1,3
  1. 1Department of Pathology, Chi-Mei Medical Center, Tainan, Taiwan
  2. 2Department of Histopathology, Addenbrooke's Hospital, University of Cambridge, Cambridge, UK
  3. 3Department of Pathology, Taipei Medical University, Taipei, Taiwan
  1. Correspondence to Dr Shih-Sung Chuang, Department of Pathology, Chi-Mei Medical Center, 901 Chung-Hwa Road, Yung-Kang City, Tainan County, Taiwan 710; cmh5301{at}mail.chimei.org.tw

Abstract

Aims Diagnosis of T-cell lymphoproliferation is sometimes challenging, and in certain instances pathologists rely heavily on the clonality assessment results of T-cell receptor (TCR) gene rearrangement (TCR-GR). Many investigators have designed various in-house primer sets for PCR-based study targeting different loci of TCR genes. In recent years, the commercial BIOMED-2 protocols have become available. The in-house primers are very cheap while the BIOMED-2 primers are expensive. This parallel study aimed to compare the sensitivity of the in-house TCRG primers (two reactions) and the BIOMED-2 TCR primers (six reactions) in an attempt to develop a sensitive and cost-effective strategy for TCR-GR assessment.

Methods PCR-based analysis was performed on 69 samples of T-lineage neoplasms including 60 formalin-fixed paraffin-embedded (FFPE) tissues, 5 samples from peripheral blood (PB) and 4 samples from bone marrow (BM) aspirate.

Results Forty-seven (78%) FFPE and all PB or BM aspirate samples yielded control DNA products suitable for clonality assessment including 4 precursor and 50 mature T-cell neoplasms. The detection rates of clonal TCR-GR were 63% (34/54) by the two in-house TCRG primers, 85% (46/54) by all six BIOMED-2 reactions, 91% (49/54) by combining the in-house and BIOMED-2 TCRG reactions and 94% (51/54) by combining the in-house and all BIOMED-2 reactions. By using the in-house and BIOMED-2 TCRG reactions with a total of four tubes, clonal TCR-GR was detected in 91% of the cases. The reagent cost for this combination was one-third of that for the six BIOMED-2 reactions and the detection rate was also higher than the latter alone (91% vs 85%).

Conclusions As the in-house primers were custom made and are much cheaper than the commercial kits, the authors concluded that this four-tube strategy was cost-effective and efficient for TCR-GR clonality assessment.

  • Clonality
  • gene rearrangement
  • non-Hodgkin's lymphoma
  • T-cell lymphoma
  • t-cell receptor
  • Taiwan
  • lymphoma
  • molecular pathology

https://doi.org/10.1136/jcp.2010.086637

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    Introduction

    During early lymphoid differentiation, genes encoding the immunoglobulin (Ig) and T-cell receptor (TCR) molecules are formed by stepwise rearrangement of variable, diversity and joining gene segments.1 2 During this recombination process, nucleotides are deleted and randomly inserted at the joining regions, resulting in an enormous diversity of lymphocytes, each carrying its own unique antigen receptor. Lymphoma cells are the progeny of a single malignantly transformed lymphocyte carrying an identical, clonally rearranged Ig and TCR genes in B-cell and T-cell lymphomas, respectively. On the other hand, heterogeneity in Ig/TCR gene rearrangement (TCR-GR) indicates polyclonal and reactive lymphoproliferations. Diagnosis of suspect lymphoproliferation is based on histopathology, immunohistochemistry and/or flow cytometric immunophenotyping with the incorporation of clinical findings. However, difficulty in differentiating benign from malignant lymphoproliferations occurs in 5–10% of cases, especially in suspect T-cell lymphoproliferations.1 2 Under such circumstances, diagnosis relies heavily on clonality study results.

    Southern blotting was the gold standard method for detecting clonality, yet it is not available in the majority of diagnostic laboratories because it requires fresh/frozen tissue and is labour-intensive. Molecular diagnosis for lymphomas has evolved from Southern blotting to the more sensitive PCR-based assay, which is widely employed in most diagnostic pathology laboratories, as it is feasible with formalin-fixed paraffin-embedded (FFPE) tissues.3–6 Given its high sensitivity of detection and short turnaround time, PCR-based clonality assays can be applied to routine diagnosis and for the confirmation of early remissions and the detection of minimal residual disease after bone marrow transplantation. Many investigators have designed various primer sets targeting different loci of the Ig and TCR genes for clonal gene rearrangement with higher detection rates (80–90%) for B-cell lymphomas, but lower (70–80%) for T-cell lymphomas.6–12 In the late 1990s, a European consortium established a highly reliable standard in PCR-based clonality testing using multiplex primers.1 Following its technical evaluation, the multiplex protocol was successfully applied to different well-defined WHO lymphoma entities with unprecedented high frequencies of malignant cases showing clonal results.1 13–17 There are currently two techniques for the analysis of PCR products obtained from Ig/TCR-GR. The heteroduplex technique is rapid, simple and cheap, and has a detection limit of ∼5%. GeneScanning is rapid and relatively simple, but needs expensive equipment. GeneScanning is generally more sensitive than heteroduplex analysis and can reach sensitivities of 0.5–1% of clonal lymphoid cells.1 Multiplex PCR-based clonality testing and assessment by GeneScan and/or heteroduplex analysis have become a worldwide standard.18–21 These protocols became commercially available as BIOMED-2.

    In our previous study published in 2003 using in-house primers targeting TCR gamma chain gene (TCRG) developed by McCarthy et al,10 we detected clonal TCR-GR in 15 of 20 (75%) cases of T-cell lymphoma.14 Since that study, we have been using these primer sets for routine diagnosis, and we began using BIOMED-2 protocols from 2007. These in-house TCRG primers are custom made and are very cheap. On the other hand, commercial BIOMED-2 TCR primers and master mixes, comprising six tubes/reactions, are very expensive in Taiwan. We performed this parallel study using in-house TCRG and commercial BIOMED-2 TCR kits on a large cohort of T-lineage neoplasms to compare their efficacy and to develop a strategy for future routine diagnoses. We found that we could detect clonal TCR-GR in 91% of cases with combined in-house and BIOMED-2 TCRG primers in a total of four reactions/tubes, a very cost-effective and efficient strategy.

    Materials and methods

    A total of randomly selected 69 samples from 67 patients with T-lineage lymphoma and/or leukaemia, both in-house and consultation cases, from 2004 to 2008 at Chi-Mei Medical Center in southern Taiwan were included in this study, which was approved by Institutional Review Board at Chi-Mei Medical Center, Tainan, Taiwan. All cases were pathologically confirmed and classified according to the 2008 WHO classification of lymphoid neoplasm.22 Fourteen cases have been reported previously in other studies.23–25 The 69 samples included 60 FFPE tissues, 5 samples from peripheral blood mononuclear cells (PBMCs) and 4 samples from bone marrow (BM) aspirate.

    For FFPE specimens, one tissue section of 5 μm thickness was deparaffinised with xylene and washed twice with 100% ethanol. DNA was extracted using the QIAamp DNA FFPE Tissue Kit (QIAGEN, Hilden, Germany) following the manufacturer's instructions. PBMC and BM aspirate samples were isolated by centrifugation with Ficoll-Paque PLUS (Amersham Biosciences, Uppsala, Sweden) and DNA was extracted using QIAamp DNA Mini Kit (QIAGEN).

    DNA quality was assessed using the BIOMED-2 control gene PCR assay. The cases with DNA product of 300 bp or more were analysed using both in-house TCRG primers and BIOMED TCR primers. Those with DNA size of 200 bp or less were considered inadequate and were excluded. The in-house TCRG primers designed by McCarthy et al consisted of two reactions comprising two forward primers (Vγ-I: TCT GG (G/A) GTC TAT TAC TGT GC and Vγ-III/IV: CTC ACA CTC (C/T) CA CTT C) and one reverse primer each of Jγ (Jγ-1/2: CAA GTG TTG TTC CAC TGC C) and JPγ(JPγ-1/2: GTT ACT ATG AGC (T/C) TA GTC C), as previously described.10 We provided the sequences to a local biotechnical company for synthesis of these primers. Commercially available BIOMED-2 multiplex PCR master mixes and controls (InVivo-Scribe Technologies, San Diego, California, USA) were used following the BIOMED-2 PCR protocols.1 The BIOMED-2 primers for TCR-GRs include two reactions targeting TCRG (TCRGA: VγI/γ10−Jγ; TCRGB: Vγ9/γ11−Jγ), three reactions targeting TCR βchain gene (TCRB; TCRBA: Vβ−Jβ1/2.2/2.6/2.7; TCRBB: Vβ−Jβ2.1/2.3/2.4/2.5; TCRBC: Dβ−Jβ) and one reaction targeting TCR δ chain gene (TCRD; Vδ−Jδ). PCR assays were run in duplicates, and polyclonal and monoclonal controls were included for each experiment. PCR products were heteroduplex-treated (denatured at 95°C for 10 min and rapidly transferred to 4°C for 60 min to promote duplex formation) and then analysed by electrophoresis on 6–8% non-denaturing polyacrylamide (19:1, acrylamide:bisacrylamide) gels in 1× Tris-boric acid-EDTA buffer, followed by staining with ethidium bromide and photographing under ultraviolet light.

    Results

    Thirteen (22%) of the 60 FFPE samples yielded control DNA products that were inadequate for clonality study (8 with 100 bp and 5 with 200 bp). Of the remaining 47 FFPE samples, 32 (68%) yielded DNA products of 300 bp, 12 (26%) of 400 bp and 3 (6%) of 600 bp. All five PBMC and four BM aspirate samples yielded DNA products of 600 bp. The 56 samples eligible for clonality study came from 54 patients including 2 patients with two samples each; their diagnoses and control PCR product sizes are listed in table 1. Specimens 17A and 17B were PBMC samples taken 15 months apart from the same patient with T-cell large granular lymphocytic leukaemia (T-LGL). Specimens 20A and 20B were BM aspirate and skin biopsy samples, respectively, taken 1 month apart from a patient with adult T-cell leukaemia/lymphoma (ATLL). The pathological diagnoses of these 54 cases included 27 (50%) cases of peripheral T-cell lymphoma, unspecified (PTCL-NOS); 10 (19%) angioimmunoblastic T-cell lymphomas (AITLs); 4 (7%) anaplastic large cell lymphomas (ALCLs); 3 (6%) T-LGLs; 2 (4%) cases each of T lymphoblastic lymphoma (T-LBL), mycosis fungoides, hepatosplenic T-cell lymphoma and mixed phenotype acute leukaemia, T/myeloid, NOS, and 1 (2%) case each of ATLL and primary cutaneous CD4-positive small/medium T-cell lymphoma.

    View this table:
    Table 1

    Case list and clonality study results for T-cell receptor gene rearrangement

    Table 1 lists the detailed results of each PCR assay. All the clonal bands fall within the expected size ranges except in case 13, in which the clonal bands by TCRG reaction was oversized. The two cases (nos 17 and 20) with two different samples showed the same clonal band patterns by using in-house TCRG and BIOMED-2 primers (figure 1). They were each considered a single case in the following analysis.

    Figure 1
    Figure 1

    Representative images from polyacrylamide gel electrophoresis of the PCR products after heteroduplex treatment of case 20 with adult T-cell leukaemia/lymphoma. The specimens were from skin biopsy and bone marrow (BM) aspirate. Both specimens show the same clonal band pattern using in-house Jγ (A), BIOMED-2 TCRBB (B), TCRGA (C) and TCRGB (D) primers. N, negative control; P, positive control; M, molecular size marker; other, the other case.

    Table 2 summarises the detection rates of in-house TCRG and BIOMED-2 reactions and their various combinations. Thirty-four (63%) cases were clonal by the in-house TCRG primers including 13 cases by Jγ primers alone, 12 by JPγ alone and 9 by both primer sets. For the six BIOMED-2 TCR tubes, the detection rates in decreasing order were TCRGA (69%), TCRGB (37%), TCRBC (31%), TCRBA (30%), TCRD (26%) and TCRBB (20%). The detection rate by the three combined TCRB tubes was 59% (32/54), while that of the two combined TCRG reactions was 78%, which was higher than the detection rate of 63% by in-house TCRG primers. The detection rates for the entire cohort were 85% (46/54) by the six BIOMED-2 reactions, 91% (49/54) by combining the in-house and BIOMED-2 TCRG reactions and 94% (51/54) by combining the in-house TCRG and all the BIOMED-2 reactions. Thirty-two (59%) of the 54 cases showed concordant TCR-GR results between in-house TCRG primers and BIOMED-2 reactions, including 29 (54%) clonal cases (case nos 1–29) and 3 (6%) polyclonal cases (case nos 30–32). Five cases (case nos 33–37) were clonal by in-house TCRG primers (2 by Jγ and 3 by JPγ) but polyclonal by BIOMED-2 reactions, while 17 cases (case nos 38–54) were clonal by BIOMED-2 reactions but polyclonal by in-house TCRG primers. Of the latter 17 cases, 15 (88%) were clonal by combined BIOMED-2 TCRB and TCRG reactions (2 by TCRB alone, 6 by TCRG alone and 9 by both TCRG and TCRB reactions). The two cases (case nos 48 and 53) clonal by TCRD reaction were also clonal by either TCRG alone or by both TCRG and TCRB.

    View this table:
    Table 2

    Detection rates of clonal T-cell receptor (TCR) gene rearrangements by in-house TCRG and BIOMED-2 reactions

    The reagent cost differs significantly between the in-house and BIOMED-2 primers in Taiwan. When calculating the reagent cost on the basis of duplicate experiments, the price for each single reaction was US$7 for in-house TCRG primers, US$65 for BIOMED-2 TCRGA, TCRGB and TCRGD and US$70 for BIOMED-2 TCRBA, TCRBB and TCRBC reactions (based on the exchange rate of 1 US$=32 New Taiwan dollars). The cost for combining the two in-house and two BIOMED-2 TCRG reactions was US$144, as compared with USD$405 when using all six BIOMED-2 primers. Furthermore, the detection rate in this study was 91% using the former combination as compared with 85% with the latter.

    We found that the most efficient and cost-effective way to detect clonal TCR-GR was using the in-house TCRG primers as the initial step with a reagent cost of US$14. The polyclonal cases would be subject to the two BIOMED-2 TCRG reactions. With this strategy, 63% (34 cases) of T-lineage neoplasms in this study were clonal at the initial screening and an additional 27% (15 cases) would be clonal by the two BIOMED-2 TCRG reactions. By combining these four reactions we could detect clonal TCR-GR in 49 (91%) of the 54 cases, higher than either in-house TCRG alone (63%) or all six BIOMED-2 TCR reactions alone (85%).

    Table 3 summarises the detection rates of clonal TCR-GR in various types of T-lineage lymphoma and/or leukaemia. All four cases of precursor T-cell neoplasm were clonal by the BIOMED-2 reactions, while only two were clonal using in-house TCRG primers. For the peripheral T-cell neoplasms, the detection rates were 64% by in-house TCRG primers and 84% by BIOMED-2 reactions. PTCL-NOS accounted for half of the study cases, and only 15 (56%) of these 27 cases were clonal using in-house TCRG primers, in contrast to 22 (81%) with BIOMED-2 reactions.

    View this table:
    Table 3

    Detection rates of clonal T-cell receptor gene rearrangements in T-lineage lymphoma and/or leukaemia

    Discussion

    Due to various factors including limited National Health Insurance coverage of molecular testing, clonality study for lymphoproliferations was rarely performed for routine diagnosis in Taiwan until recently. We have previously used the same in-house TCRG primers for a smaller series of T-cell lymphomas and detected clonal TCR-GR in 75% (15/20) of cases.12 In our current study, the detection rate by the in-house TCRG primers (63%) is lower than our previous series. If we pooled all cases in these two studies together, the detection rate would be 66% (49/74), comparable with or slightly lower than those reported in the literature.6–12 These in-house primers were initially developed by McCarthy et al, and their detection rate was 70% using snap frozen tissue from 14 cases of T-cell leukaemia and 6 cases of PTCL.10 In this protocol, there are no primers covering the VγII family (Vγ9) genes, which might partly explain the lower detection rate than that of the other protocols (up to 80%) covering this gene family.1 9 11 However, in the current study we detected five clonal cases by these in-house TCRG primers which were undetectable by the BIOMED-2 primers, indicating that these in-house primers are valuable adjuncts to BIOMED-2 protocols for TCR clonality study.

    The first PCR-based clonality testing using BIOMED-2 primers in Taiwan has recently been published.26 In that study, Chen et al used custom made primers synthesised according to the sequences published in the original paper by van Dongen et al1 and detected clonal TCR-GR in 72% (18/25) of T-lineage lymphomas.26 In our study, the detection rate using commercial BIOMED-2 kits is higher than that obtained in the study by Chen et al (85% vs 72%).26 One of the reasons for their lower detection rate might be that they used custom made reagents in place of the commercial BIOMED-2 kit, which might have been ‘refined’ or ‘fine-tuned’ with a better sensitivity. When we started using BIOMED-2 protocols a few years ago, we used the same approach as Chen et al and obtained similar suboptimal results. Subsequently, we sought to compare the efficacy between the commercial kits and the custom made BIOMED-2 primers, and found that some clonal cases detected by the former were undetectable by the latter, even after stringent purification of the primers and adjusting the concentrations of magnesium chloride and other chemicals (unpublished observation). After this initial phase, we chose to use the commercial BIOMED-2 kit for this study.

    Clonality analysis is best performed on high molecular weight DNA extracted from fresh/frozen specimens as recommended by the College of American Pathologists.27 However, in routine haematopathological practice, paraffin-embedded tissues are often the main type of specimens available for molecular investigations and DNA extracted from such tissues are extensively degraded. To enable clonality analysis applicable to such materials, BIOMED-2 protocols were developed, with the majority of the primers designed to generate products of less than 300 bp.1 The DNA quality is checked by control DNA gene PCR and those samples yielding amplified control PCR products of 300 bp or over were considered suitable for clonality assessment. In the original BIOMED-2 paper, DNA was extracted by using the QIAamp DNA Mini Kit (QIAGEN); 21/45 (47%) FFPE samples yielded control DNA products of 200 bp or less.1 Such a high proportion of samples with inadequate DNA quality hamper the routine diagnostic application of clonality testing. During the initial phase of this study, we switched from this extraction kit to the newer QIAamp DNA FFPE Tissue Kit (QIAGEN) and obtained DNA products of a better quality from the same FFPE samples of many patients (data not shown). In this current study, 22% of FFPE samples yielded control DNA products of 200 bp or less. A similar level of failure rate has been recently reported by others using similar18 28 (21% of 43 and 28% of 40 FFPE samples, respectively) or different29 (21% of 316 FFPE specimens) protocols for DNA extraction. Therefore, it is reasonable to expect a similarly high failure rate for FFPE haematopathological specimens in general practice. Further improvement of DNA quality will require change of the practice for specimen handling. Prolonged fixation, use of non-neutral buffered formalin or other fixatives such as B5, Bouin's and acids, and long specimen storage are known to cause excessive DNA fragmentation.1 27 A recent audit in one of the authors' (HL) institutions showed that over 95% of freshly prepared FFPE specimens yielded DNA products of 300 bp or over following introduction of a more strict protocol for fixatives (10% neutral buffered formalin) and fixation time (overnight for surgical resections and shorter, usually 3–8 h, for small biopsies) for all histology specimens (unpublished data). It seems that adequate but not prolonged fixation in 10% neutral buffered formalin would result in a significant improvement in nucleotide preservation in fixed tissues.

    From our case series, the combined use of two in-house TCRG tubes and two BIOMED-2 TCRG tubes, that is, the four-tube strategy, will detect a clonal TCR gene rearrangement in 91% of cases with a confirmed diagnosis of T-cell neoplasm with adequate DNA quality. Performing other BIOMED-2 reactions is needed only for the remaining 9% of cases, of which 3% are likely to show a clonal TCRB and/or TCRD gene rearrangement. Such an overall level of T-cell clonality detection rate compares well with the 94% detection rate reported in the follow-up study of the BIOMED-2 group13 and the 93–96% detection rate reported in the independent studies using BIOMED-2 assays combined with laboratory developed assays on fresh-frozen materials.30 31 Thus, approximately 5% of false negative results are expected in T-cell malignancies with adequate DNA quality. The causes for the false negative results are unclear. Incomplete coverage of the rearranged TCR gene segments by the primers designed or unusual rearrangement of TCR genes may be a factor.1 A small subset of T-cell neoplasms such as AITL and ALCL are known to lack detectable clonal TCR-GR.13 For cases with amplifiable DNA products of less than 300 bp, it is worth noting that such samples may still be amenable for analysis with both in-house and BIOMED-2 TCRG assays, in particular the former, as the size ranges of PCR products of Vγ−Jγ and Vγ−JPγ are small (64–100 bp and 80–110 bp, respectively), and thus are more suitable to degraded DNA. Nevertheless, extra caution is required when interpreting clonality results in such specimens.

    In a comparative PCR-based study using in-house TCRG and TCRB primers for both paraffin and frozen tissues, Diss et al showed that monoclonality detected by the former primers was higher than the latter (78% vs 44%) and concluded that TCRG PCR should be the method of choice for TCR clonality analysis in FFPE specimens as TCRB PCR has a lower sensitivity and a higher false negative rate.11 In our study, the detection rate using BIOMED-2 TCRB and TCRG primers were 59% and 78%, respectively, and 83% when combining both tests. Our results were similar to two recent studies using the same kits on paraffin-embedded skin tissue with primary cutaneous T-cell lymphomas including mostly mycosis fungoides.32 33 In these studies, the addition of BIOMED-2 TCRB primers slightly increased the detection rate. In our study, among the 17 polyclonal cases by in-house TCRG reactions, 15 were clonal by combining BIOMED-2 TCRG and TCRB reactions and 2 additional clonal cases were identified by TCRB reactions alone. In some previous studies,13 34 BIOMED-2 TCRD reactions were reported to have a low detection rate with little added value. Similarly, we found that no additional clonal cases could be identified by BIOMED-2 TCRD reaction alone after using TCRB and TCRG reactions.

    Diagnosis of T-cell neoplasms sometimes relies heavily on the demonstration of immunophenotypic aberrancy and T-cell clonality. Aberrant T-cell immunophenotype and/or monoclonal TCR-GR usually indicate clonal and malignant T-cell lymphoproliferation. However, it is very important for pathologists to bear in mind that while in most cases monoclonality confirms malignancy, clonal T-cell populations can be found in reactive conditions, such as inflammation and infection.1 35 36 For example, we had encountered a paediatric patient with a massive expansion of Epstein–Barr virus-positive monoclonal T cells with downregulated CD5 expression and haemophagocytic lymphohistiocytosis in the BM.37 Furthermore, detection of oligoclonal and/or clonal proliferation of T cells is common in older people and is considered as a sign of ageing of the immune system rather than neoplastic lymphoproliferation.38 The results of clonality studies should be interpreted in the context of the clinical, morphological and immunophenotyping findings to reach a correct diagnosis.

    In summary, we found from this parallel study that PCR-based clonality assessment using in-house TCRG primers was an adjunct to the BIOMED-2 protocols for detecting TCR-GR. By combining the in-house and BIOMED-2 TCRG reactions with a total of four tubes, we could achieve a relatively high detection rate (91%) at a relatively low cost. As the in-house primers could be custom made and are much cheaper than the commercial BIOMED-2 kits, we concluded that this four-tube strategy was cost-effective and efficient for TCR clonality assessment.

    Take-home messages

    • T-cell receptor gene rearrangement (TCR-GR) assessment is sometimes very important for the diagnosis of suspect T-cell lymphoproliferation.

    • Our in-house TCRG primers for TCR-GR comprising two reactions were cheap with a detection rate of 60–70%. On the other hand, the commercial BIOMED-2 TCR primers with a total of six tubes were expensive with a sensitivity of up to 85–90%.

    • In-house TCRG primers are supplementary to the BIOMED-2 primers for TCR-GR. Our four-tube strategy (two in-house and two BIOMED-2 TCRG reactions) is cost-effective and efficient for TCR-GR clonality assessment.

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    Footnotes

    • Funding This study was supported by research grants from Chi-Mei Medical Center (CMFHR9913), Tainan, and National Science Council (97-2320-B-384-001-MY3), Taipei, Taiwan.

    • Competing interests None.

    • Ethics approval This study was conducted with the approval of the Chi-Mei Medical Center, Tainan, Taiwan.

    • Provenance and peer review Not commissioned; externally peer reviewed.