Tag-deleted SV40-derived vectors (rSV40s), since they do not elicit neutralizing antibody responses, and so can be given multiply without loss of transduction efficiency.A vaccine that elicits durable, powerful anti-HIV immunity remains an elusive goal. In these studies we tested whether multiple treatments with viral vector-delivered HIV envelope antigen (gp120), with and without IL-15, could help to approach that goal. For this purpose, we used recombinant SV(gp120) carried the coding sequences for HIV-1NL4-3 Env, and SV(mIL-15) carried the cDNA for mouse IL-15. Singly, and in combination, these two vectors were given monthly to BALB/cJ mice. Cytotoxic immunity and cytotoxic memory were tested in direct cytotoxicity assays using unselected effector cells. Antibody vs. gp120 was measured in a binding assay. In both cases, targets were P815 cells that were stably transfected with gp120.Multiple injections of SV(gp120) elicited powerful anti-gp120 cytolytic activity (>70% specific lysis) by unselected spleen cells. Cells from multiply-immunized mice that were rested 1 year after their last injections still showed >60% gp120-specific lysis. Anti-gp120 antibody was first detected after 2 monthly injections of SV(gp120) and remained elevated thereafter. Adding SV(mIL-15) to the immunization regimen dramatically accelerated the development of memory cytolytic responses, with ≥ 50% specific lysis seen 1 month after two treatments. IL-15 did not alter the development of antibody responses.Thus, rSV40s encoding antigens and immunostimulatory cytokines may be useful tools for priming and/or boosting immune responses against HIV. The development of an effective vaccine against HIV has been hindered by a variety of problems. The high mutation rate of the virus itself is such that it represents a moving antigenic target during the course of an infection -4. FurthCompared to administration of protein antigen or naked DNA, an infectious vector could be more effective at enhancing antibody and cytotoxic responses against a transgene product. Application of such a strategy, however, has been often complicated by the development of neutralizing immune responses, principally antibodies, against vector coat antigens -10. ThesTag-deleted SV40-derived gene delivery vectors (rSV40s) for immunization. Several studies have shown, both directly and indirectly, that rSV40 vectors do not elicit detectable neutralizing antibodies , to yield prSVmIL-15. Virus was made from this plasmid in COS-7 cells as previously reported  minus [c.p.m. 51Cr released by SV(HBS)-immune lymphocytes from gp120-expressing P815 cells]}, divided by [c.p.m. 51Cr released by Triton X-100 from gp120-expressing P815 cells]. The same calculations were done for lysis of wild type P815 cells by gp120-immune and control-immune effector populations. These numbers were then subtracted from the calculated 51Cr release above to determine the gp120-specific lysis of target cells by SV(gp120)-immunized effector cells.% specific P815 cells were transduced with SV(mIL-15) ×1 at m.o.i. = 100. Culture supernatants were harvested at several times post-transduction, and stored at -80°C. At day 6 post-transduction, a well of cells was harvested and lysed ). Remaining wells were activated non-specifically with 5 mg/ml Con A, and supernatants harvested at various times thereafter. 3 days after con A stimulation, cells were lysed as described above. 50 μg of each culture supernatant or lysate were loaded on a 4–20% Tris-HCl gradient gels . 50 ng recombinant human IL-15 was used as a positive control. Samples were electrophoresed, and blotted to PVDF membranes . Blots were blocked overnight at 4°C with 5% milk in PBS + Tween-20 (0.05%). Rabbit anti-mouse IL-15  was used as primary antibody, (diluted 1:500 with PBS-Tween), for 2 h at 37°C. Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG  was used at 1:10,000 dilution in PBS-Tween, for 1 h at room temperature. Signal was detected with chemiluminescence reagent COS-1 cells were infected with SV(mIL15) or SVLUC (carrying luciferase) as a negative control. 24 h later, the media were changed and cells incubated 48 h in 500 μl of RPMI 10% serum/well. Fresh mouse spleen cells (5000/well) then cultured 48 h with 100 μl of cell supernatant + 100 μl of RPMI 10% serum. IFNγ ELISA (Pharmingen) was performed on the supernatant from these cultures.Methods). We used flow cytometry to identify the clone most strongly positive for cell membrane gp120. Compared to other stably-transfected clones, "clone 24" expressed gp120 at the cell membrane best (data not shown). VCB41-infected and SV(gp120)-transduced P815 cells also expressed substantial cell membrane gp120. Control wtP815 cells, or P815 cells infected with wt VV did not (data not shown).P815 cells stably transfected to express HIVNL4-3 gp120 were selected and cloned by limiting dilution )-immunized rSV40-immunized mice vs. clone 24 cells. Thus, data presented below reflect gp120-specific responses against clone 24.Methods. Specific binding antibody activity was first statistically significant, compared to prebleed sera 2 weeks after the second inoculation of SV(gp120)  and reached a plateau after the third inoculation  , and their sera were assayed for reactivity vs. gp120 by CELISA. The details of this cell-based ELISA, or CELISA, as described in 20, and tAn effective anti-lentiviral immunization regimen should generate cytotoxic memory cells. To see if SVgp120) treatment could do this, mice were immunized once with SV(gp120) IP, then sacrificed 1 month later, without further treatment. In order to lyse target cells, committed cytotoxic cells require activation. However, to avoid antigen-specific selection and specific stimulation only of gp120-reactive cytotoxic cells, splenic lymphocytes were non-specifically stimulated by overnight incubation with Con A. A single immunization with SV(gp120) alone elicited only weak memory lytic responses (≤ 10% specific lysis) against gp120-expressing target cells  one month after the first, then were assayed the same way one month later for anti-gp120 cytolytic activity. These mice made stronger specific memory responses (15–20% specific lysis) than did animals given only a single inoculation   gp120-specific cytolytic responses (mean specific lysis of 61% ± 4.2).To test whether SV(gp120) could elicit very long term cytotoxic lymphocyte memory, mice were immunized monthly ×8 with SV(gp120) IP. A final IP inoculation with SV(gp120) was given Methods, using rabbit antibody vs. murine IL-15 , and lower levels were seen with 2 injections, we tried to accelerate development of such responses using IL-15, delivered by transduction. To determine if IL-15 could be expressed by transduction, P815 cells were transduced with SV(mIL-15) at m.o.i. = 100. Culture supernatants were harvested 36, 72 and 144 hours later, at which point the cultured cells were activated with Con A. Culture supernatants were collected at 24 and 72 hours post-activation. Supernatants were assayed for IL-15 secretion by Western analysis as described in The functionality of the IL-15 produced in this fashion was tested by exploiting the ability of IL-15 to elicit production of IFN-γ by lymphocytes. Thus, CV-1 and COS-7 cells (African green monkey kidney cells) were transduced by SV(mIL-15), then cultured for 72 hrs. Control cultures of the same cells were transduced with SVLUC (carrying luciferase as a transgene). Normal mouse spleen cells were cultured for 42 hrs in 200 μl of the resulting culture supernatants. Production of IFN-γ by the spleen cells was measured by ELISA. Supernatants from COS-7 and CV-1 cells elicited respectively 2056 ± 363 pg/ml and 880 ± 196 pg/ml IFN-γ. Supernatants from SVLUC-transduced cells did not elicit detectable interferon secretion by spleen cells (<20 pg/ml).9 IU of rSV40: one group was given SV(mIL-15) alone, and one group SV(gp120) alone. Three other groups received both SV(mIL-15) and SV(gp120): either SV(mIL-15) followed 3 days later by SV(gp120), or SV(gp120) first, followed by SV(mIL-15). The final group was given both SV(mIL-15) and SV(gp120) simultaneously. The 3 day separation between the two vectors was used because of the strength of the signal for secreted IL-15 by Western blotting at 72 hours post-stimulation (see above). One month later, unselected spleen cells were assayed as described above for cytolytic activity against gp120-expressing clone 24 cells.To determine whether coordinate administration of SV(mIL-15) plus SV(gp120) improved cytolytic responses against gp120, mice were given two sets of injections IP, one month apart. Normal BALB/c mice received IP with 10Adding IL-15 to the immunization regimen greatly increased gp120-specific cytolytic responses Figure . Also, tMethods.Mice receiving SV(mIL-15) and/or SV(gp120) ) according to the schedules outlined above were tested to determine the effect, if any, of such co-administration in anti-gp120 serum antibody responses. CELISA and calculation of gp120-specific antibody binding were performed as described in Slight binding antibody activity was detected 2 weeks after the first inoculation(s), in all SV(gp120) recipient groups. Levels of SV(gp120)-induced groups made detectable antibody responses were not appreciably affected by coadministration of SV(mIL-15) (data not shown).in vivo. Our results here demonstrate several important strengths of using rSV40 vectors to immunize against lentiviral antigens: Among these are the ability of the vector to be administered multiple times without eliciting neutralizing responses [51Cr-release assays: unselected lymphoid organ populations were added directly to labeled target cells at low E:T ratios, and specific 51Cr release was measured.In this study, we used rSV40 vectors to elicit HIV-1NL4-3 gp120-specific cytotoxic lymphocyte and antibody responses. We have observed that these vectors may be administered repeatedly to boost those responses. Further studies also suggested that such responses are durable esponses -13, and 51Cr release observed in the current studies were extensively controlled to ascertain the antigen-specificity of the cytolysis observed: background lysis of wild type P815 cells was subtracted, as was lysis by lymphocytes from mice immunized with an irrelevant rSV40 vector. We also found that cytolysis increased with increasing numbers of immunizations, which is not a characteristic of NK cell-mediated lysis.Analysis to confirm CD8 expression, or expression of other CTL markers was not performed on the effector cells. However, it is unlikely that these data reflect the cytotoxic activity of NK cells. NK cytolytic activity is non-specific and does not increase with repeated immunization. The patterns of in vivo, we tested whether cytotoxic lymphocyte activity elicited by SV(gp120) immunization, was detectable one month after inoculation. Thus, cytolytic responses, assayed one month after a second injection, were ≈ 20%, which is comparable to those of splenic cytotoxic cells assayed four days following a third inoculation (data not shown). Further, mice given multiple injections of SV(gp120), then rested for one year, gave ≈ 70% specific lysis when challenged with SV(gp120). Therefore, SV(gp120) administration may thus favor development of cytotoxic lymphocyte memory.Since a key goal for a vaccine against HIV is to generate immune responses that are durable In an attempt to accelerate and to improve upon these specific cytotoxic and particularly cytotoxic memory responses, we co-immunized mice with SV(gp120) and a rSV40 carrying mouse IL-15. IL-15 promotes cytotoxic lymphocyte responses, and in particular, cytotoxic memory responses ,24. The in vitro. Furthermore, low effector:target ratios (20:1 and 10:1) were used in these assays. Our immunization protocols tested both simultaneous and staggered administration of rSV40s carrying HIV-1NL4-3 gp120 and murine IL-15.Accordingly, our analysis of the contribution by IL-15 to cytotoxic responses, focused mainly on the ability of SV(mIL-15) to augment specific cytotoxic responses of spleen cells from animals rested 1 month following immunization. Because quiescent cytotoxic T cells are not strong effectors, we non-specifically activated the splenocytes prior to assay with Con A. Non-specific activation was used to avoid specifically enriching effector cell population for gp120-specific cells IL-15 co-immunization dramatically accelerated cytotoxic responses, depending on the immunization regimen used: Animals given SV(mIL-15) alone made no gp120-specific cytolytic responses. Mice receiving 2 treatments with a mixture of SV(gp120) and SV(mIL-15) gave much higher specific lysis, depending on the coadministration regimen, as compared to those receiving SV(gp120) alone (≈ 10% specific lysis). Thus, among mice given staggered injections of SV(mIL-15) and SV(gp120), the order of cytokine administration greatly affected the response: if SV(gp120)was given first, no detectable gp120-specific cytolysis was observed. However, if the cytokine was given first, followed 3 days later by SV(gp120), ≥ 60% specific lysis was seen at both 20:1 and 10:1 effector:target ratios.Why the order of cytokine administration should affect antigen-specific responses so dramatically is not yet clear. Cytokine given after, or together with antigen, may have insufficient time to augment cytotoxic responses. In addition, Western analysis of IL-15 production showed that IL-15 secretion was not detectable in supernatants beyond 36 hours, but could be stimulated subsequently. Thus, a specific, possibly brief, window for IL-15 expression and secretion may need to be attained, in order for its effects on gp120-specific responses to be detectable. We observed very high levels of specific lysis by these unselected effector populations following just two tandem injections of SV(mIL-15) followed by SV(gp120).The strong anti-lentiviral cytolytic responses we report were observed in a strain of mouse, BALB/cJ, that generally mounts relatively weak type 1 T cell responses. The finding of >60% cytolysis with two administrations of SV(gp120) + SV(mIL-15), suggests that a strategy similar to that described herein may be helpful in individuals who would generate relatively low cytolytic responses.Serum antibody levels assayed by CELISA where SV(gp120) was administered alone, multiple times, were detectable after two immunizations, and continued to increase up to week 4 following the third immunization. These responses were not further enhanced by subsequent boosting immunizations. While specific antibody responses against gp120 were detected in all experimental groups following SV(gp120) and SV(mIL-15) co-immunization, IL-15 co-administration did not augment anti-gp120 antibody levels, compared to gp120 alone. This was to be expected, since IL-15 reportedly acts primarily on T cell and NK cell functions, rather than on humoral immune responses.Our data argue in favor of using IL-15 as an adjuvant for antigen-specific immune responses, particularly cytotoxic lymphocyte responses. We also demonstrate that a single transgene, administered multiple times (>3), may be very effective at eliciting both humoral and cell-mediated responses. These results thus both corroborate and extend our previous observations ,14,32, aRecombinant SV40-derived gene-delivery vectors, being transparent to the immune system, can be given multiple times to prime and boost immune responses against the delivered antigens. Anti-vector immunity does not overwhelm responses against the target antigens. As well, these vectors elicit very high levels of antibody, and especially cell-meditated immunity. Finally, combining the delivery of rSV40s bearing antigens with those bearing cytokines such as IL-15 can enhance levels of immunity, particularly long-term immunity. Clearly, much work remains. However, this approach offers promise as a strategy to immunize against pathogens for which classical approaches have not been adequately effective.None declared.HJM devised all the assay systems for cell- and antibody-mediated immunity against lentiviral antigens, performed all the immunization studies and assays. HM also wrote this manuscript. PYT generated the SV(gp120) construct. MV and PF generated the SV(mIL-15) and SVLUC constructs described here and performed the ELISA for IFNγ stimulated by SV(mIL-15). DSS is the Principal Investigator for this work, oversaw and planned the experimental strategies, worked with HJM in interpreting the experimental data and writing the manuscript.


Homo sapiens, followed by sampling humans from different ethnic backgrounds, and chimpanzees, we have identified 27 NUMTs that are specific to humans and must have colonized human chromosomes in the last 4–6 million years. Thus, we measured the fixation rate of NUMTs in the human genome. Six such NUMTs show insertion polymorphism and provide a useful set of DNA markers for human population genetics. We also found that during recent human evolution, Chromosomes 18 and Y have been more susceptible to colonization by NUMTs. Surprisingly, 23 out of 27 human-specific NUMTs are inserted in known or predicted genes, mainly in introns. Some individuals carry a NUMT insertion in a tumor-suppressor gene and in a putative angiogenesis inhibitor. Therefore in humans, but not in yeast, NUMT integrations preferentially target coding or regulatory sequences. This is indeed the case for novel insertions associated with human diseases and those driven by environmental insults. We thus propose a mutagenic phenomenon that may be responsible for a variety of genetic diseases in humans and suggest that genetic or environmental factors that increase the frequency of chromosome breaks provide the impetus for the continued colonization of the human genome by mitochondrial DNA.Integration of mitochondrial DNA fragments into nuclear chromosomes  is an ongoing process that shapes nuclear genomes. In yeast this process depends on double-strand-break repair. Since NUMTs lack amplification and specific integration mechanisms, they represent the prototype of exogenous insertions in the nucleus. From sequence analysis of the genome of  DNA from mitochondria has regularly inserted into the human nuclear genome. Some insertions are polymorphic, revealing that the invasion of the human genome is an ongoing process Insertion of new sequences into nuclear DNA has a major impact on its architecture and is an important mechanism for the evolution of eukaryotic genomes. Moreover, when targeted to gene loci, these insertions can be mutagenic, and in humans this process contributes to a number of diseases . The freDNA fragments of mitochondrial origin, originating from both coding and noncoding regions, are found as sequence fossils in the nuclear genomes of various eukaryotes . HoweverHomo sapiens, experimental approaches that compare individuals within this species and its closest relative, the chimpanzee, can be undertaken .Our updated analysis (data not shown) reveals that the majority of these NUMTs correspond to those previously documented . HoweverTo determine whether sequences of mitochondrial origin were actually integrated in the human nuclear genome, and were not a result of contamination of DNA library preparations, we selected 42 NUMTs for analysis in human samples. Our choice included the 36 NUMTs with the highest identity (91% to 100%) to the mtDNA, one NUMT having the longest stretch of DNA with high identity (88%), one NUMT corresponding to the highly variable region of the mtDNA (D-loop) , and fouIn summary, results from two amplification strategies and from sequencing demonstrated that these NUMTs were indeed present at the expected chromosomal location and that they are bona fide mt sequences residing in the human nuclear genome.The colonization of human populations by various NUMTs revealed striking disparities. Thirty-five NUMTs were present in homozygous form in all individuals tested see . InteresInterestingly, one or more of these six NUMTs were detected among individuals within each ethnic group, indicating that their insertion in the nuclear genome occurred soon after the origin of modern humans and that they represent the most recent integrations of our studied cases. Despite the limited sampling size , the frequency of alleles carrying the insertion varies greatly according to the NUMT . For each locus, one to three chimpanzee individuals were analyzed. Forty-two out of 42 primer pairs successfully amplified the target site also in chimpanzees because of the high sequence identity of the two genomes (average 98.7%)  are unlikely to be human-specific. This results in an average of one NUMT integration in the germline for each 180,000 y, in the last 4–6 million years (Myr). Interestingly, one fourth of these NUMTs  show insertion polymorphism  . Only the 98.7%) . All of The distribution of human-specific NUMTs in human chromosomes is not proportional either to the chromosome size or to the total number of NUMTs present in the chromosome . In ChroQ8N7L5 into two, and the NUMT itself becomes a new intron , a species closely related to humans and whose evolutionary relationship with humans has been widely investigated, is an ideal candidate for a comparative analysis. Moreover, the high level of identity (more than 98%) between the two species (H. sapiens. For NUMTs fixed in the human genome (not displaying insertion polymorphism), we do not expect this value to increase significantly, since our analysis was made on essentially the entire human genome.An important question concerning the integration of DNA sequences in the nuclear genome is their rate of colonization. For exogenous sequences like NUMTs, this has not been investigated in vivo. To determine the extent of colonization of a given genome, it is necessary to compare the insertions within this genome with those of a closely related species. To date, a comprehensive analysis of the presence of NUMTs has been done for several complete nuclear genomes , but the species  allows tH. sapiens and have integrated in the human genome in the last 4–6 Myr, after the split of the two species from their common ancestor (Homo erectus out of Africa (1.7 Myr ago). Living humans would still be polymorphic for these NUMTs, as a result of interbreeding of the nonmodern human populations with modern humans  are specific to ancestor . This coancestor . Howevern humans . On the n humans . In thisCompared to 28 NUMT insertions in the human nuclear genome in the last 4–6 Myr, it has been calculated that about 5,000 new insertion events of Alu repeats have occurred in the human genome in the same timescale  “BLAST the Human Genome” server (http://www.ncbi.nlm.nih.gov/genome/seq/page.cgi?F=HsBlast.html&&ORG=Hs). The blastn program was used with default parameters on April 24, 2003. Only output parameters were changed to 1,000 descriptive lines and to 1,000 segment alignments. In a few cases  were obtained as purified DNA from Coriell Institute . Purified chimpanzee DNA, obtained either from tissues or from fecal material, was a kind gift from J.-P. Vartanian at the Pasteur Institute . For both PCR strategies described in the text, primer sequences are available upon request. Cell lysis was performed by incubating fresh cells overnight at 55 °C in a Tris-EDTA buffer (pH 8.5) in the presence of 200 μg/ml of proteinase K. PCR amplification was performed with 30 cycles of denaturation (1′ at 94 °C), annealing (1′ at 68 °C), and DNA synthesis (3′ at 72 °C) using Invitrogen  −8 mutations per nucleotide per generation, or 1.25 × 10−9 mutations per nucleotide per year, assuming a generation time of 20 y , where d is the frequency of sequence divergence between the NUMT and present mtDNA sequence. As an example, for a sequence 300 bp long, 94% identity to mtDNA corresponds approximately to an insertion time of 3.3 Myr, and 96% to 2.2 Myr.The age of insertion of NUMTs was estimated using, as reference, the sequence divergence of the NUMT from the mtDNA. We assumed that the NUMT, when inserted into the nuclear genome was identical to the corresponding mt sequence. We also assumed that, once inserted into the nuclear genome, the NUMT mutated at the same rate as the nuclear genome, μN, which corresponds, for noncoding sequences, to 2.5 × 10 of 20 y . By compe D-loop . Thus, fTable S1(53 KB DOC).Click here for additional data file.Table S2(63 KB DOC).Click here for additional data file.http://www.ncbi.nlm.nih.gov/genome/seq/page.cgi?F=HsBlast.html&&ORG=Hs) accession number for the human mtDNA sequence is AB055387.The NCBI (


Extramedullary myelomas (plasmacytoma) are malignant proliferations of plasma cells in the absence of bone involvement. When they occur in the soft tissue they usually involve the upper respiratory tract and oral cavity. Extramedullary plasmacytomas of breast are uncommon.A 70 year-old woman with bilateral breast masses underwent excisional biopsy for suspected primary carcinoma that subsequently proved to be a recurrence from extramedullary plasmacytoma of the mediastinum. This was diagnosed and treated 5-years prior to appearance of breast lumps.Though uncommon, considering the possibility of metastatic carcinoma and primary, secondary or recurrent lymphoproliferative disease presenting as a breast mass may avoid unnecessary surgeries. Extramedullary myelomas (plasmacytoma) are malignant proliferations of plasma cells in the absence of bone involvement. When occur in the soft tissue, it usually involve the upper respiratory tract and oral cavity . PlasmacThis report describes a patient with bilateral breast masses who underwent excision biopsy for suspected primary carcinoma that subsequently proved to be a recurrence from extramedullary plasmacytoma of mediastinum treated 5 years ago. To the best of our knowledge, this is the first case report of bilateral recurrence of a primary extramedullary plasmacytoma in breast tissues after a long disease-free interval.A 70 year-old woman with a one-month history of bilateral breast masses was referred to our cancer center for surgical evaluation. There was no associated breast pain, skin change or nipple discharge. There was no history of bone pain, weight loss, fatigue, fever or other systemic complaints and no family history of breast cancer. Significant past medical history included treatment for an extramedullary retrosternal plasmacytoma 5-years prior this admission.At the time of the initial work-up for the retrosternal mass, immunoelectrophoresis showed no evidence for hyperproteinemia or paraproteinemia. Whole body bone scan was negative and a bone marrow biopsy revealed less than 5% of plasma cells. Therefore, multiple myeloma was excluded by nuclear medicine, laboratory and histology studies. The patient underwent radiation therapy (40 Gy with fraction size of 200 cGy delivered over 4 weeks) followed by chemotherapy with cyclophosphamide, cisplatin and prednisolone. The patient was followed by laboratory tests, chest roentgenography, and computed tomography annually. A bone scintigraphy was carried out after 2 years and showed no uptake patient was thereafter lost to follow-up.Five years after initial diagnosis of extramedullary plasmacytoma the patient presented with bilateral breast masses. Physical examination revealed a 3.5 cm × 2.5 cm, mass in the upper inner quadrant of the right breast and a similar 5.0 cm × 4.5 cm mass in the lower inner quadrant of the left breast. No asymmetry, skin dimpling or signs of inflammation were present. There was no axillary or supraclavicular lymphadenopathy.Mammography confirmed a well-defined 3.2 cm oval-shaped mass in the upper inner quadrant of the right breast, and a lobulated 5.5 cm density in lower inner quadrant of the left breast without any tissue distortion, inflammation and fibrotic reaction.Figure  There we99 bone scan and skull and pelvic X-rays) did not show any pathological changes. There was no evidence of anemia, hypercalcemia or renal insufficiency. However, the patient refused a second bone marrow biopsy.Excisional biopsy of the masses revealed a 5.0 (left) and 3.0 (right) well-defined, capsulated gritty mass surrounded by normal breast tissue. There was no extension from the capsulated masses to pectoral muscles or chest wall. Histopathological examination showed high-grade tumors composed of immature and mature plasma cells. Mitosis, necrosis, nuclear pleomorphism and binucleated and multinucleated plasma cells were seen. Figure  AdditionImmunohistochemical studies were performed on the paraffin embedded tissues to determine if the infiltrate had monoclonal character. The tumor cells were diffusely and strongly positive for lambda chains but negative for kappa chains. Figure The tumor cells were weakly positive for monoclonal mouse anti human placental V538C, and plasma cell markers (CD138). Nuclear prognostic marker (Ki67) showed 50% to 80% nuclear expression indicative of high proliferative activity and suggesting a plasmacytic tumor with anaplastic components Figure . Other iA retrospective microscopic review of the mediastinal mass showed similar morphology to the breast tumor. Hence, the histological diagnosis of recurrent plasmacytoma was made.The patient was treated with oral Melfalan and Prednisone. The patient has been disease free for twenty months after treatment and has showed no evidence of recurrence in the mediastinum, breast or any other region.Primary soft tissue extramedullary plasmacytoma (SEP) is uncommon and is defined as a malignant tumor of plasma cells arising in the soft tissue in the absence of bone involvement. It can occur in any organ as a solitary form of plasma cell neoplasm . AlthougWhen plasmacytoma originates from soft tissues, like the case presented here, the disease is usually associated with a relatively mild clinical behavior and long survival, suggesting that it is a truly different disease entity compared to other plasma cell tumors .et al (1999) reported that solitary extramedullary (soft tissue) plasmacytomas (SEP) are less common than solitary bone plasmocytoma (SBP), and have a better prognosis as the majority can be cured by local radiotherapy [Dimopoulos otherapy .et al (1999) reported local recurrence rates of less than 5% after radiotherapy [et al (1990) noted that the risk of distant relapse is more than 30%, which is significantly less than that seen with SBP [Liebross otherapy . Mayr etwith SBP . Progreswith SBP .Involvement of the breast with SEP is uncommon and may occur either as a solitary primary tumor or as evidence of disseminated multiple myeloma . Our patOur patient did not have any prior breast aspiration cytology. There are many tumors, which may present with plasmacytoid appearance on aspiration cytology. Tumors such as small ductal carcinoma and lobular carcinoma of breast, metastatic carcinoid, metastatic melanoma and some lymphomas may represent with uncohesive group of cells with eccentric nuclei resembling plasma cells. Hence, a plasmacytoma (with anaplastic plasma cells) may be readily mistaken for carcinoma (or other undifferentiated neoplasm) not only clinically, but also on cytological examination. This would justify excision biopsy and the use of an extended immunohistochemical panel to include such markers as cytokeratin and S-100 in the assessments. To help the cytopathologist avoid misinterpretation, clinical history and presentation are extremely helpful. Most of the errors in histopathology and cytopathology diagnosis occur when pathologist is not aware of medical history of the patient and unusual clinical presentation.This case emphasizes the importance of distinguishing a plasmacytoma of the breast from primary mammary carcinomas and other benign lesions to avoid unnecessary surgery and provide the appropriate treatment and adjuvant therapy.AK carried out excision biopsies and drafted the manuscript.MJZ did the histopathological examination and contributed to the pathological content of the manuscript.SKR is the pathologist who confirmed the diagnosis and prepared the immunohistochemical stains and illustrations. He also contributed to pathological content of the manuscriptMN followed-up the patient and contributed to the manuscript preparationAll authors read and approved the final manuscript.


Quantitative proteomics is an emerging field that encompasses multiplexed measurement of many known proteins in groups of experimental samples in order to identify differences between groups. Antibody arrays are a novel technology that is increasingly being used for quantitative proteomics studies due to highly multiplexed content, scalability, matrix flexibility and economy of sample consumption. Key applications of antibody arrays in quantitative proteomics studies are identification of novel diagnostic assays, biomarker discovery in trials of new drugs, and validation of qualitative proteomics discoveries. These applications require performance benchmarking, standardization and specification.Six dual-antibody, sandwich immunoassay arrays that measure 170 serum or plasma proteins were developed and experimental procedures refined in more than thirty quantitative proteomics studies. This report provides detailed information and specification for manufacture, qualification, assay automation, performance, assay validation and data processing for antibody arrays in large scale quantitative proteomics studies.The present report describes development of first generation standards for antibody arrays in quantitative proteomics. Specifically, it describes the requirements of a comprehensive validation program to identify and minimize antibody cross reaction under highly multiplexed conditions; provides the rationale for the application of standardized statistical approaches to manage the data output of highly replicated assays; defines design requirements for controls to normalize sample replicate measurements; emphasizes the importance of stringent quality control testing of reagents and antibody microarrays; recommends the use of real-time monitors to evaluate sensitivity, dynamic range and platform precision; and presents survey procedures to reveal the significance of biomarker findings. Traditional immunoassay platforms have very limited multiplexing capability and high sample volume requirement. The development and application of high throughput, multiplex immunoassays that measure hundreds of known proteins in complex biological matrices, is becoming a significant tool for quantitative proteomics studies, diagnostic discovery and biomarker-assisted drug development [reviewed in -4]. Two Unique, general considerations in assembling multiplexed immunoassays include: Requirements for elimination of assay cross-reactivity; configuration of multi-analyte sensitivities; achievement of dynamic ranges appropriate for biological relevance when performed in diverse matrices and biological states; and optimization of reagent manufacturing and chip production to achieve acceptable reproducibility. In contrast to traditional monoplex enzyme-linked immunoassays, generally agreed specifications and standards for antibody microarrays have not yet been formulated. A number of recent articles have started to examine certain of these issues ,6,7.Microarray immunoassays performed on planar glass slides and employing signal enhancement with rolling circle amplification (RCA), have been developed by several groups and have demonstrated usefulness in measurements of temporal and dose-dependent changes in a variety of immunological model systems and human diseases ,2,8-16; -16; 8-16I. Analytes in an applied sample bind to capture antibodies immobilized on a silanized glass surface.II. Applied secondary biotinylated detector antibodies bind to captured analytes, creating a highly specific immune complex.III. Biotinylated detector antibodies bound to the immune complex are detected with a universal anti-biotin antibody. The latter is conjugated to primer oligonucleotides that are pre-annealed to a complementary circular oligonucleotide.IV. DNA polymerase extends the 3' ends of primers around the circles, resulting in long, single stranded RCA products that remain attached to the complex.The RCA product, composed of tandem DNA repeats complementary to the circle sequence, is detected by hybridization with cyanine 5 (Cy5)-labeled complementary oligonucleotides.The present report describes initial development of standardized operating procedures, quality controls and standards for microarray immunoassays performed on planar glass slides using signal enhancement with RCA. These metrics have been tested for use in generation of data with adequate sensitivity, reproducibility and assay performance for biomarker discovery -14,16, P, P16, PaTo demonstrate the feasibility of using a multiplex immunoassay system to measure protein levels in complex biological matrices, the performance of dual-antibody, sandwich immunoassay arrays performed on planar glass slides with RCA signal enhancement was evaluated for specificity, sensitivity, reproducibility and accuracy using standardized titrations, spiked biological matrices and clinical samples. Array performance was evaluated based on ability to: measure analytes across a broad dynamic range at sufficiently low coefficients of variation (CVs); detect proteins at levels requisite to capture biologically relevant expression differences; confirm reliability of methods to normalize data to minimize platform imprecision and demonstrate the utility of generating standard curves to convert analyte MFI (mean fluorescence intensity) data into mass unit information.An advantage of arrays is the ability to measure each analyte multiple times, enhancing precision. Capture antibody spots were printed in quadruplicate on planar glass slides providing redundancy of individual analyte measurements. Data redaction was applied to raw immunoassay data to improve data quality by eliminating outlier data points. Outliers were identified by employing two subsequent statistical approaches in a step-wise manner.First, the Bland-Altman plot was used. Bland-Altman plots are often used in DNA microarray analysis to identify differences and/or replicate outliers. This involves plotting the difference between the logarithm of intensities of two replicates (M) versus the average of logarithm of intensities (A) for each analyte within an individual array . Thus, there will be 6 MvA plots for each data set to reflect the 170 analytes positioned across 6 arrays. Each MvA plot will contain 3*Ns*Na points, where 3 reflects the number of possible unique pair wise combinations of the three replicates, Ns represents the number of samples and Na defines the number of analytes measured on a given array. An example of an MvA plot produced in a project comprising 150 clinical serum samples for Array 4 with 37 analytes is shown in Figure 2) is examined for each plot. Each plot contains Na data points, where Na reflects the number of analytes. Plots with R2 values <0.95 are examined to identify the cause of the poor correlation. We have identified two major sources of poor correlation: incorrect positioning of the capture grid during image quantitation and general aberrations in image spot quality. Assigning the specific source of low correlation is accomplished by tracing back to the image data. In the case of grid misplacement, suspect data images are re-quantified. Poor correlations due to aberrant spot morphology/intensity are manually examined and removed from data set. If the R2 value does not improve as a result of outlier removal, the replicate is redacted from the data set. The sample is considered passed if there are two replicates with R2 >= 0.95. The pass rate is defined as the number of passed samples divided by the total number of samples. A run of an array is considered to be passed if 85% of the samples have two or more passed replicates.The second step of data reduction involves a linear correlation analysis. Pair-wise correlation analysis is done between all replicates of individual sample. Figure 2 values < 0.95. This process reduces throughput of data redaction, particularly on large data sets. Table In our experience, applying the MvA statistical approach first, followed by the linear correlation analysis is an efficient process to identify outliers without compromising data throughput. Since, MvA plots can be generated quickly, it allows for relatively fast redaction of significant outliers using an objective semi-automated approach. In contrast, the sample correlation analysis is considerably more labor intensive and currently requires manual investigation of each scatter plot with RIn general, for data sets with more than 40 samples, outlier removal only demonstrated small improvements in reducing average CVs across all samples. The most significant impact of outlier removal is on improving reproducibility across the three replicates of the individual samples. In our experience, outlier removal has been shown to reduce individual sample replicate CVs by 2–3 fold. This effect is directly related to improving sample correlation pass rates by by 10–20%.Many systematic factors can modify spot intensity during the process of measurement. Normalization is the process of reducing the effects of systematic variation on spot intensity. Normalization in DNA microarrays typically involves adjusting distributional summaries of data  from each chip to common reference values. For example, one assumption could be that the average signal from each protein chip should be the same, as with DNA microarrays and the difference between replicate values is due to systematic variability in the measurement process. Unfortunately, the nature of protein antibody microarrays, configured with a multiplex of individual capture and detector antibodies, is more specialized and differentiated than that of a DNA microarray. Use of a single reference factor derived from a global value is not sufficiently refined to take into account the difference in platform configuration. In the current report, the organization of protein microarrays allows the measurement of up to 16 samples per slide (chip). This is very different from DNA microarrays where one chip represents the total collection of measured values for an individual sample.To accommodate the differences inherent to the platform, we have applied a normalization strategy based on the three major sources of technical variability observed in our system. The first type of variability relates to spot-to-spot differences observed between quadruplicate spots of the individual analytes printed within a sample well. The second level of variability can be described as the difference in measurements between wells within the same slide. The third element of variability represents the variability observed between sample wells compared across different slides. We found that slide-to-slide variability is the largest source of variation accounting for more than 70% of the total measurement imprecision (data not shown). Thus the goal of normalization is to reduce the imprecision of slide-to-slide measurement error since this represents the major source of platform variability.Normalization is performed using a system of standard controls to reduce the effect of slide-to-slide variability. A series of four standard control samples  are run in 4 wells of each slide. Each control sample represents a cocktail of the full repertoire of analytes for the given array tittered at 4 specific concentrations. The standards have been optimized at concentrations  to capture measurements across the linear range of detection for each analyte. The global average of total analyte signal for the four prepared controls is calculated across all slides run in a batch. An adjustment factor is created for each slide that reflects the difference between global intensity average for all slides and the individual intensity average based on the controls from the individual slide. The averaged pixel intensity of each spot on the slide is scaled by the adjustment factor.As an example, the average value of the adjustment factor was evaluated across a batch of 33 slides and found to have a value of 1.33+/- 0.47. The primary benefit of normalization was in reducing the replicate sample CVs. Figure A 15-point series of standardized titrations containing recombinant proteins diluted in buffer were used to evaluate platform precision. This assessment was used in the quality control of each slide lot prior to release, as well as within each client project to verify run-time analyte performance. Six replicates for each point were run in the quality control testing of each slide lot and six replicates of each point were run within each client study to generate standard curves. CVs were evaluated for each concentration of analyte across six slides. Average CVs were calculated for each analyte. Statistical summaries of CV distribution across all array 2 analytes using the standardized 15-point standard titration series are shown in Table A variance decomposition analysis was performed to reveal the extent to which platform error influenced the ability to identify biomarkers. The variance component assigned to platform error was typically found to be an order of magnitude lower than the average inter-individual variation. Figure The left panel of Figure A performance assessment of individual analytes was conducted to determine the utility of each analyte across multiple projects covering diverse disease areas. Each analyte was evaluated according to the percentage of clinical samples that fell within (W), below (B) or above (A) the linear range of detection. Tables ,7,8,9,10The development of an antibody array featuring 25–40 novel immunoassays requires extensive validation related to the comprehensive assessment of antibody cross reactivity, definition of analyte minimal detection limits (MDL) and establishing robust assay performance. Each antibody array must be validated for use with several matrices, since the latter may have different ambient analyte levels  or cross-reactivity profiles.Analyte sensitivity was assessed to identify analytes lacking adequate performance for retention on an array. Additional experiments were performed to determine the endogenous levels of each analyte. For analytes without previously reported biological values, the "0 × n" assays indicated the approximate ambient analyte level. Testing across multiple biological matrices was required, since different matrices affected the detection of analyte specific signals. The "0 × n" experiments also revealed the level of non-specific background which was influenced by the total concentration of antibody load in the detector mix. In our experience, certain plasma matrices were also more likely to generate high background when compared to matched serum samples. The impact of high generalized background is a reduced sample pass rate. When background was observed, the total detector antibody concentration could often be reduced to minimize background noise. Ultimately, a balance between reduction in background and enhancement of sensitivity was required to achieve maximal analyte performance in a mutiplex configuration.The results of the 1 × (n-1) assays identified analytes that demonstrated cross-reaction between the captured analyte and the complex detector mix prepared without the cognate detector antibody. Binding between the spiked analyte and the cognate capture that generated signal, indicated a cross-reaction to one or more non-cognate detector antibodies contained within the complex mix. In cases where non-cognate detector signal was observed, an additional series of experiments were run with the corresponding analyte tested against each of the individual detectors to identify the cross-reacting detector antibody. Since cross reaction is an additive process, the outcome of the cross reaction assessment allowed for adjustments to be made to achieve a balance between maximizing content with multiplexed array specificity. The 0 × (n-1) assays were run to provide a baseline of MFI values to compare to the results obtained in the 1 × (n-1). In addition, the 0 × (n-1) experiments also served to screen the various biological matrices for cross-reactivity with endogenous proteins.2 values obtained between the two conditions provided a measurement of the accuracy of the multiplexed system.Serum MDLs were typically found to be higher than buffer MDLs due to the presence of endogenous analyte, potential analyte-binding proteins present in the biological matrix and other possible matrix-related interferences. The assay conditions used to stress test the system under conditions of high analyte load were designed to identify cross-reaction thresholds for each of the individual analytes. MFI cut off values were used to identify significant increases in non-cognate signal that warranted removal of a feature from the array. The results provide a certain utility in predicting array performance under conditions where sample analyte concentrations exceed reported biological levels. Examples might include patient samples tested under diseased states, elevated analytes produced in stimulated cell culture supernatants or in samples exhibiting a strong drug response. The final validation involved measuring the accuracy of the multiplex assay when challenged with a high concentration of analyte. Figure Thirty years of widespread use of conventional, monoplex immunoassays has established firm benchmarks for performance in protein measurement. In the present paper, we have examined several, unique but general considerations in assembling multiplexed immunoassays with performance similar to conventional monoplex immunoassays. These include development of a comprehensive validation program to identify and minimize antibody cross reaction under highly multiplexed conditions; application of standardized statistical approaches for data handling for highly replicated assays; inclusion of standardized samples in each run to normalize sample replicate measurements; quality control of reagents and antibody microarrays; implementation of real-time monitors to evaluate sensitivity, dynamic range and platform precision; and initial procedures for identification of specific, significant immunoassay results in biomarker discovery projects involving clinical samples. Each of these will be discussed briefly.An array validation program represents the foundation of tests required to establish robust assay performance in a multiplexed environment. The most significant component in array validation is the comprehensive evaluation of cross reactivity. The vast majority of the ~5000 commonly available antibody pairs available today have not previously been evaluated for cross reactivity in a multiplexed environment. Therefore, the recommended program should include procedures that identify analytes demonstrating cross reactivity with immobilized capture antibodies as well as cross reaction that might manifest between the secondary detector antibody with a non-cognate analyte or non-specific binding to an immobilized capture agent.The performance of analytes in a multiplexed configuration should be benchmarked against the baseline, monoplex performance. This multiplexed immunoassay comparison with baseline performance, together with minimal standards for multiplexed cross-reactivity, permits determination of the practical, optimal number of array elements that can be successfully combined. In our experience, using dual-antibody, sandwich immunoassays, planar glass slides and RCA signal amplification, protein micorarrays can generally accommodate multiplexing of 25–35 analytes without an appreciable drop in individual analyte sensitivity or performance. Specifically, we have described development of six different dual-antibody sandwich immunoassay arrays, each containing 25–37 sandwich immunoassays. Since cross reactivity is an additive process, the ultimate goal is to achieve a balance between maximal multiplexing and monoplex-like performance. With exhaustive selection for antibodies without cross-reactivity in multiplexed format, it is possible to multiplex 50 sandwich immunoassays. However, this exercise is very expensive. In our experience, suspension arrays, alternative microarray surface substrates and attachment chemistries do not offer significant advantages in multiplexing while maintaining performance. We have not evaluated the impact of novel, affinity ligands on multiplexing.Additional array validation for cross-reactivity should include "stress-testing" under high analyte load to reflect conditions where analytes may be significantly over-expressed. In our experience, levels of induction of proteins in common biological matrices can be very large following drug administration or in disease states, and may induce cross-reactivity that is not observed in testing within normal biological analyte levels.Finally, array validation should be performed across all common sample matrices to examine effects on assay performance associated with endogenous analyte, matrix specific analyte binding proteins or other matrix-specific inhibitors. Absence of cross-reactivity for an immunoassay in one matrix does not always imply absence of cross-reactivity in others. The matrices for which the antibody microarrays described herein have been validated include isotonic buffers, serum, citrate plasma, heparin plasma, EDTA plasma, cell culture supernatants, amniotic fluid, sputum, and exhaled breath condensates. Several of the arrays have also been validated for use with ex vivo treated whole blood. EDTA plasma and ex vivo treated whole blood had higher levels of background signal and lower sample pass rates than other matrices.2> 0.95 range were eliminated. In order for data from sample replicates to pass and be admitted into the final data set, the overall sample replicate-to-replicate correlation for the 25–37 analytes of the array was required to have an R2> 0.95. Experience in multiplexed immunoassay measurements in samples across more than 30 research projects indicated the R2 value >0.95 to be routinely achievable and associated with high quality replicate data. In each project, the data lost through these two sequential redaction procedures was typically less than 5% of the total original data. An additional quality metric to assess the overall run performance was that at least two of the three replicates must have passed for 85% of the total samples. Runs falling short of this metric were failed and subject to repeat. The typical run fail rate was less than 3%.A significant advantage of array-based immunoassays is the ability to measure each analyte in a sample many times. Removal of outlier replicates is obligatory for microarray assays due to signal-related and morphology-based artefacts typically associated with dispersing small volumes of material on a solid substrate in a microarray format. Application of standardized statistical approaches for data redaction is superior to manual inspection and removal of outliers since operator-dependent subjectivity is minimized and throughput is greatly increased. The data redaction procedures described herein employed two, separate steps: Bland-Altman plots and linear correlation analysis. Bland-Altman plots were employed first and identified 99% confidence intervals for all collected data points. This enabled rapid identification and elimination of the outlying 1% of the data with minimal human intervention. This was determined to be an objective, reproducible redaction procedure that greatly reduced time and effort associated with the subsequent, second data redaction step of linear correlation analysis. Linear correlation analysis required performance of 3 replicate assays on each sample, and manual inspection of the series of 3 scatter plots generated from pair-wise correlations of these 3 sample replicates. Individual replicate points for each specific analyte that fell outside the RWithin-run controls were employed to account for the effects of systematic variation in replicate measurements. Variation was identified at three levels based on the unique configuration of the 16 sample well microarray chip. The lowest level of variability was observed between the quadruplicate spots of an individual analyte measured within a single sample well. The next level of variation was described as the difference between replicate analyte values measured in different wells located within the same slide. The highest level of variation was associated with measurements taken from a single sample applied to multiple wells positioned across different slides within a run. Since slide-to-slide variation demonstrated the highest system variation, a series of four controls were designed to minimize the impact on sample replicate measurements. The four controls contained all analytes for that array at four concentrations spread across the dynamic range. The four controls were run on every slide within a project and used to generate a global average of total analyte signal. Based on the global average, each individual slide was assigned an adjustment factor to compensate for the slide specific intensity bias. The analyte signal from each individual slide could then be scaled by the adjustment factor to normalize the intensity values between the sample replicates positioned across different slides. In addition, it is possible to use a blocking experimental design, intentionally positioning sample replicates across different slides and different slide locations to eliminate the potential for a slide-specific or location-specific intensity bias. An example of the latter might have been the well at the corner of a slide. Replicate measurements in conjunction with a mechanism to normalize systemic variation results in the production of high quality data required for maximal sensitivity in the identification of significant differences between samples in multiplexed immunoassays. An additional benefit of inclusion of standardized controls run across all slides of every project is the ability to standardize data, for example in mass units, and enable data comparisons between runs, between days and between projects. Such comparisons are necessary when projects constitute large numbers of samples or when it is desired to create a relational database of assay results. Our platform described herein, for example, can perform triplicate measurements on up to 200 samples in a single run.Quality control of approximately 1200 individual reagents is necessary in order to provide consistent performance of 170 immunoassays on the array platform described herein. These reagents, unfortunately, have widely different shelf life and storage conditions. Stringent quality control procedures specifying performance metrics associated with these reagents were required to achieve reproducible array performance across hundreds of slide lots and reagent sets. Each new lot of a given component was benchmarked to an earlier lot to verify performance. Analyte intensity, dose-response curve, LLD/ULD absolute values, dynamic range and background signal were evaluated in fuctional tests performed on all assay components. Historical performance was monitored by comparing running averages obtained from earlier lots to prevent performance change over time. These procedures were made practical by assembling cocktails of reagents for each step in an assay, dispensing these in single-use aliquots, establishing optimal storage conditions and shelf life, and performing regular  quality checks on aliquots. Implementation of such procedures required use of a laboratory information management system.The utility of integrating real-time platform performance monitors cannot be understated. Given the complex nature and potential instability of biological reagents associated with a multiplex antibody array, it is critical have a program in place to evaluate performance beyond the quality control release. Real time monitors measure performance of controls under conditions identical to the test samples and reflect a second level verification of assay performance. Our test system employed a series of monitors to capture precision metrics that would create a flag to review the data if the specifications were not met. The requirements included mean coefficients of variation of assay values for controls be less than 15% and for sample replicates be less than 25% for project samples run within a batch. Failure to achieve these metrics indicated a problem related to the performance of the manufactured slides and/or reagents or a technical failure associated with sample handling or assay execution. 15-point standardized titrations were also performed on 6 slides in every run in order to captured detail related to analyte dynamic range, LLQ/ULQ values, dose response behaviour, and background signal that provided a comprehensive assessment of real time platform performance. The detail of the performance assessment was included in final reports for each project to verify data quality and generate confidence in the data generated from a highly complex assay.Evaluating data generated from multiplexed immunoassays for utility in systematic identification of significant differences between samples, or "biomarker discovery", is an important step in understanding the true platform performance. One of the procedures that revealed the sensitivity of the platform for biomarker discovery was variance decomposition analysis for each project. Variance decomposition analysis examines the magnitude of individual components of platform variation and how they compare to analyte variation between samples or individuals. In our experience the platform error of the system described herein was generally an order of magnitude lower than the heterogeneity observed between samples or individuals of the same test group. The utility of this test is in revealing the extent to which platform error impacts the ability to discover moderate expression level differences between samples that are reflective of biological change. Platforms with lower precision will have less sensitivity for detection of relevant differences between samples and will discovery only a subset of the markers that would have been identified with a more precise system.Finally, a global performance assessment should be performed across multiple projects covering diverse disease areas to gain a solid understanding of the platform utility. An evaluation of this type can be used to identify assays that will not identify differences in expression between samples because they are not sufficiently sensitive, unable to generate sufficient dynamic range given the window of expression, or reveal high endogenous abundance producing assay saturation artefacts. In addition, specific assays that have appropriate sensitivity and dynamic range may be constitutively expressed and therefore poor biomarker candidate analytes for certain disease or treatment effect studies. This analysis may be used to direct efforts to continue to optimize the survey platform in order to generate the highest value in identifying biomarkers using a quantitative proteomic approach.Protein microarrays offer the ability to simultaneously survey multiple protein markers in an effort to develop expression profile changes across multiple protein analytes for potential use in diagnosis, prognosis, and measurement of therapeutic efficacy. The current report details certain minimal standards, use of which was found to be necessary to generate the requisite specificity, sensitivity and reproducibility to discover biomarkers. Results revealed that a multiplex system could be operated with high analyte specificity, adequate detection sensitivity and sufficiently broad dynamic range to capture expression differences across diverse disease and therapeutic areas.Raw soda-lime glass slides (1" × 3") prepared with a Teflon mask configured to provide 16 individual sample wells and an etched barcode for traceability were subjected to visual inspection to identify imperfections that might translate into printing and/or scanning artifacts. Slides with scratches, surface contamination or defects in the applied Teflon mask were identified through a visual examination using a long wavelength inspection lamp equipped with a 532 nm filter. The inspection also failed slides that did not meet stringent dimensional specifications, required for downstream printing and automated assay conditions.Slides passing the visual inspection were silanized with 3-cyanopropyltriethoxysilane according to procedures previously described . MeasureCapture antibodies prepared as previously described  were priEach printed array contained 256 spots representing 64 individual elements printed in quadruplicate. Each array contained 25–40 capture antibodies spread across production chips 1–6, generating a panel of 170 survey analytes. The balance of the elements was reserved for internal assay controls. Each printed array contained multiple copies of an element called BLANK, containing the components used in capture antibody preparation. Blanks were used to survey non-specific sample background within each well. Other printed controls included a series of biotinylated mouse IgG calibration standards to monitor RCA signal amplification and a third control that acted as a monitor for spot contamination resulting from carry-over between sequentially printed features.Preparations of standardized multiplex analyte titration series were manufactured using recombinant analytes diluted in buffer that covered the range from 12 pg/mL up to 81 ng/mL at 14 discrete points along with two zero analyte buffer blanks. These titration points were distributed among the sixteen available wells on a slide . Manual immersion washes were substituted with pipette delivered solutions finely tuned to control pipette tip aspiration and delivery position above printed slide wells and to carefully control liquid application and aspiration speeds to minimize disruption to the assembled immunosandwich complex. Incubation times were increased from 30 to 45 minutes for two of the assay steps  and the number and volume of washes between steps increased from 2 to 4–5 and from 20 uL to 30 uL respectively. A Tecan LS200 unit was used to scan the slides. Microarray images were quantified using image capture software (ImageGrabber) developed in-house.Frozen serum samples from over 800 clinical patients were thawed, centrifuged to remove particulate matter and mixed with 0.25 mg/ml Heteroblock (Omega), 0.25 mg/ml IIR (Bioreclamation) and 0.1% Tween-20 prior to the assay. Twenty microliters of serum was applied to each well.2(Rep1/Rep2) versus the average of the log intensities (A=log2((Rep1*Rep2))/2) for each of the replicates across all analytes. Patterns were visualized using fitted curves from robust local regression with applied visual cues to identify a 99% confidence interval. All outliers in the MvA plot outside of the interval  were automatically removed from analysis. The MvA scatter plots also allowed the user to highlight subsets of points on the plot and investigate patterns of intensity differences observed between replicate values. In cases where redaction of an entire replicate  was too stringent, individual spots could be removed using an in-house developed software tool (Terminator) to visually inspect aberrant data points. Data redaction using this method was performed on a limited basis to remove individual spot outliers with poor circularity, non-uniform pixel intensity or missing spots.Data points producing outlier events as a result of missing spots, spots with poor morphology, or printed features demonstrating high pixel outliers were removed using a combination of automated and manual methods. MvA plots, were generated by plotting the difference of the log intensities (M=log2) value cutoff. An ideal microarray, when compared to its identical replicate, would have a R2 value of 1. Any comparison producing values lower than the cutoff would result in at least one failed replicate. Individual sample correlations were generated by plotting analyte MFI values  from each replicate against the other replicates individually covering all combinations of replicates over the 25–37 analytes within the array. The R2 values obtained for the three plots were manually reviewed to identify failed sample replicates. Only sample replicates with R2 values >0.95 for replicates run within a day or R2 values >0.90 for replicates run across multiple days passed the correlation QC. A summary of the overall sample replicate pass rate monitored the number of failed replicates observed across each of the individual arrays. Project performance specifications required that >85% of all study samples had at least 2 reported replicates.As a quantitative QC measure, data review included a sample replicate correlation assessment with a predefined correlation coefficient  scale to generate within-slide titration curves. Linear regression coefficients (slope and intercept) were calculated between individual titration curves from each slide to generate an "average" titration curve. Calculated slope and intercept were used to transform averaged analyte values for each sample well. Data normalization was performed on the data set after outlier removal.A standardized precision assessment was performed on each run to monitor assay performance with respect to; within well variation (based on mean coefficient of variation (CV) observed between quadruplicate printed spots for all features across all sample wells of a project) and between-slide variation . The mean and median CVs with standard deviations were also metrics included in the precision assessment. The precision assessment was performed as a quality control using the 15-point titration calibrators to qualify new slide lots and generate quality metrics for each client project.Mean fluorescent intensity (MFI) values, on a logarithmic scale, from the 3 replicate measurements of the 15 point standard titration series were used to generate precision profiles to define the upper and lower limit of quantitation  within a predefined concentration CV . To do tThe VARCOMP procedure of SAS (SAS Institute), was used to obtain estimates of the variance components in a mixed model. The fixed effect variable represents variance observed in different groups in the study, for example groups of healthy versus diseased individuals. Random effects were represented by unique sample identifiers nested within levels of a fixed variable. This component of variance represented within-group differences associated with patient-to-patient variability or disease heterogeneity. The residual variance represented the platform error.Assay sensitivity was determined in two series of experiments. Initial testing to identify analyte cross-reactivity was performed in a configuration where all printed capture antibodies are surveyed in a "1 × n" format, representing a single recombinant analyte tested against all (n) detectors across multiple matrices . Capture antibodies that revealed binding to non-cognate antigens were removed or replaced with suitable alternatives. Analytes that demonstrated low signal across all matrices were removed. If signals were low in buffer, a comparison was made with signals obtained in serum or plasma to determine if the endogenous analyte level was detectable and determine if depressed signals were due to analyte instability in a non-biological matrix. Assessment of analyte endogenous level was performed using a "0 × n" format where unadulterated serum and plasma  are assayed with the full complement of detector antibodies for a given array.Two conditions were examined to study potential cross-reactivity between the complex detector antibody mixture and the immobilized capture analyte. The first condition included a 1 × (n-1) format in which 1 analyte was tested in the presence of all detectors minus the detector antibody specific to the added analyte (n-1). In the case of an array containing 40 printed features, 40 unique detector antibody cocktails are prepared containing 39 of the detector antibodies found in the complex mix, with each mix containing all but one of the 40 corresponding detectors. The 40 individual reaction mixes are added to specific arrays after the arrays were incubated with the antigen corresponding to the missing detector. The second condition represented the 0 × (n-1) format where no analyte was added in the presence of all detectors minus the detector specific to the analyte under examination. In each case the analytes were spiked in buffer, serum, and plasma  at a fixed analyte concentration of 50 ng/ml.Single analyte titrations were prepared in buffer, serum and plasma  to assign a minimum detection limit (MDL) for each analyte based on a 95% confidence interval above background. The format of the experiments included an n × n design, where all analytes were run in the presence of all detectors. Then, using a 1 × n format, where only one antigen was added to an assay containing all detectors (n), each analyte was tested at 0X, 10X, 50X, and 100X MDL across the same test matrices to identify non-cognate cross-reaction under high analyte load. Additional antigen titration experiments were run to compare the performance achieved in the presence of a single antigen (1 × n) to one in which all analytes were present (n × n).MFI: Mean Fluorescence Intensity.RCA: Rolling Circle Amplification.CV: Coefficient of Variation2: correlation coefficientRLLQ: lower limit of quantitationULQ: upper limit of quantitationMDL: minimal detection limitsSD: standard deviationL.T. Perlee, J. Christiansen, B. Grimwade, S. Lejnine, V. Tchernev and M. Sorette were employees of Molecular Staging, Inc. and D.D. Patel and S.F. Kingsmore have received consulting fees.RD oversaw the manufacturing of reagents, JC established standardized quality control testing procedures, MS implemented assay automation and oversaw all clinical testing. BG was responsible for data curation, SL performed statistical analysis. MM and WS contributed to array develoment. SFK, VTT and DDP were involved in study design, LTP in project execution and LTP, SL, JC and SFK in manuscript preparation.