We aimed to determine optimal strategies for complete mortality ascertainment comparing death certificates and United States (US) Veterans Administration (VA) records.We constructed a cohort of California veterans who died in fiscal year (FY) 2000 and used VA services the year before death. We determined decedent status using California death certificates linked to VA utilization data and the VA Beneficiary Identification and Records Locator System (BIRLS) death file. We compared the characteristics of decedents who would not have been identified by either single source  with the rest of the cohort.A total of 8,813 veteran decedents were identified from both VA decedent files and death certificates. Of all decedents, 5,698 / 8,813 (65%) veterans were identified in both source files, but 2,426 / 8,813 (28%) decedents were not identified in VA BIRLS, and 689 / 8,813 (8%) were not identified in death certificates. Compared to the rest of the cohort, decedents whose mortality status was ascertained through either single source differed by race / ethnicity, marital status, and California residence. Clinically, veterans identified from either single source had less comorbidity and were less likely to have been users of VA inpatient or long term care, but equally or more likely to have been users of VA outpatient services.As single sources, VA decedent files and death certificates each provided an incomplete record, and death ascertainment was improved by using both source files. Potential bias may vary depending on analytic interest. Clinicians, healthcare administrators, researchers, regulators and policymakers are concerned with optimizing mortality ascertainment using administrative data. In addition to its clinical importance, mortality informs program planning, quality assessment and improvement, and public reporting -8. VeterIn order to evaluate the implications for improving veterans' end-of-life care, we constructed a population-based decedent cohort . For sucThe VA Beneficiary Identification and Records Locator System (BIRLS) contains records of all beneficiaries including veterans whose survivors applied for burial benefits. It includes records of discharged military veterans post-1973 and recipients of Medals of Honor and VA education benefits. After submission to the Veterans Benefits Administration (VBA), deaths are recorded in the BIRLS Death File. A submission to the VBA is typically triggered by a family claim for death benefits  -19. The We first identified 345,380 decedent veterans who died during FY2000 (30 September 1999 – 1 October 2000) from the BIRLS Death File. We used SSNs to link cases to VA NPCD outpatient, inpatient, or long term care records restricted to recipients of any VA services in California within 12 months of death. We extracted records including any inpatient or long term care admission, or outpatient encounters. Veterans who entered the cohort on the basis of using outpatient services were required to have at least one clinical encounter (e.g., other than laboratory, radiology, or administrative).In addition, we used California death certificates as second source to identify decedent veterans by linking SSNs from death certificates directly to VA utilization files. California death certificates contained 462,561 records for calendar years 1999 and 2000, and we primarily matched decedents identified through death certificates to BIRLS by SSN. We manually inspected matches on SSN only and we also examined matches on criteria other than SSN (e.g. last name, first name, date of birth, date of death). Additional cases we accepted after manual inspection involved transpositions of one and rarely more than one SSN digit but agreement in other fields. Thus, the cohort included recipients of VA clinical services verified as deceased based on either BIRLS or death certificates, and all cases were linked to VA utilization files by SSN.In the final decedent cohort, we excluded cases of non-veterans receiving care at VA facilities by examining indicators of veteran status associated with visits. The VA assigns specific codes to non-veterans rendered care for various reasons (e.g., emergency or charitable care). We also considered the possibility of erroneous decedent status by looking for evidence of healthcare utilization during the 12 months after death. We excluded cases with evidence of utilization more than one month after the date of death.We used VA encounters and ICD-9-CM codes to demographically  and clinically characterize decedents -26. We iTo understand the limitations of single source mortality ascertainment, we described decedents who would not have been identified by a strategy using either death certificates alone or VA decedent files alone. We compared these cases with the rest of the cohort based on their demographic and clinical attributes and the settings in which they received care. Based on distributions, we used Wilcoxon tests for continuous and chi-square tests for categorical variables.From 345,380 deaths during the period 30 September 1999 to 1 October 2000 identified in BIRLS, we distinguished 6,071 decedents who were users of VA inpatient, outpatient, or long term care services in California. California death certificates included 227,308 deaths during the same period, including 3,580 additional users of VA inpatient, outpatient, or long term care services in California. Using SSN and other identifiers to match decedent cases to VA utilization data, we excluded non-veterans (n = 365), users of only non-clinical care such as laboratory tests (n = 251), those possibly alive based on subsequent VA encounter data (n = 229), and 3 cases for other reasons. Of the final cohort of 8,813 veteran decedents, 5,698 (65%) cases were identified in both source files, while 689 (8%) were only identified in VA decedent files, and 2,426 (28%) additional cases were only identified through death certificates . Ninety-nine percent of decedents missed by using VA data alone were California residents (vs. 92% of the remainder cohort, p < 0.001); whereas, 62% of those missed by using death certificates alone were out-of-state residents (vs. 1% of the remainder cohort, p < 0.001). Relatively fewer veterans of white or black ethnicity and relatively more veterans of missing ethnicity were represented among decedents missed by either single source strategy. The proportion of married or previously married veterans was higher and single or missing marital status lower among those decedents missed using only BIRLS, and relative proportions were reversed for a strategy using only death certificates.Decedents missed by either single source approach were less likely to have been diagnosed with an advanced chronic illness than the identified cohort. Veteran decedents missed by using only BIRLS were less likely to be diagnosed with any condition except HIV and dementia, and those missed by using death certificates alone were less likely to be diagnosed with any condition except HIV. With a BIRLS only approach, 37% of missing cases vs. 35% of the remainder cohort p < 0.001) had no diagnosed chronic illness . Veteran decedents missed by either single source approach were equally or more likely to have been users of the outpatient setting, but missed cases were less likely to have been users of inpatient healthcare settings  were both significantly lower among veterans missed by either single source approach.Our findings are consistent with Washington State where the deaths of 25% of 533 veterans who only used outpatient services were only identified with death certificates, and 5% were only identified in BIRLS.  Using BIOne limitation of our study is that we did not identify cases that were only decedents by virtue of VA utilization files alone rather than BIRLS, although Dominitz, et. al., identified only 2.7% of deaths this way . We did Researchers, managers, and policy makers should understand the limitations of sources of mortality ascertainment. The relationship of missing data to bias is related somewhat to how "missingness" is distributed by the outcome of interest. Our findings suggest these concerns may be relatively more important for studies involving veterans and racial-ethnic disparities, co-morbidity, certain disease comparisons, or settings of care. Additional study is needed to compare BIRLS, death certificates, and the NDI for mortality ascertainment in veterans. If our findings are confirmed, the VA may need to consider improving its system for mortality ascertainment through routine linkages to national mortality data. Studies of end-of-life care using decedent cohorts need to pay particular attention to the incompleteness of VA data as the sole source of mortality information.VA, Veterans Administration; FY, fiscal year; BIRLS, Beneficiary Identification and Records Locator System; NPCD, National Patient Care Database; SSN, Social Security Number; CHF, congestive heart failure; COPD, chronic obstructive lung disease; ESLD, end-stage liver disease; ESRD, end-stage renal disease.The author(s) declare that they have no competing interests.KL originated and oversaw all aspects of the conception, design, analysis, and publication of the study. SA, LR, and EY contributed to conception, design, and analysis. MW contributed to analysis and is responsible for programming. All authors reviewed and approved of the manuscript.


Small quantities of RNA  available from biological samples frequently require a single round of amplification prior to analysis, but current amplification strategies have limitations that may restrict their usefulness in downstream genomic applications. The Eberwine amplification method has been extensively validated but is limited by its ability to produce only antisense RNA. Alternatives lack extensive validation and are often confounded by problems with bias or yield attributable to their greater biological and technical complexity.To overcome these limitations, we have developed a straightforward and robust protocol for amplification of RNA in the sense orientation. This protocol is based upon Eberwine's method but incorporates elements of more recent amplification techniques while avoiding their complexities. Our technique yields greater than 100-fold amplification, generates long transcript, and produces mRNA that is well suited for use with microarray applications. Microarrays performed with RNA amplified using this protocol demonstrate minimal amplification bias and high reproducibility.The protocol we describe here is readily adaptable for the production of sense or antisense, labeled or unlabeled RNA from intact or partially-degraded prokaryotic or eukaryotic total RNA. The method outperforms several commercial RNA amplification kits and can be used in conjunction with a variety of microarray platforms, such as cDNA arrays, oligonucleotide arrays, and Affymetrix GeneChip™ arrays. Here, we present an overview of RNA amplification strategies, identify key limitations to existing techniques, and describe a simple, robust, and cost-effective strategy for single round amplification of RNA in the sense orientation.The increased use of microarray expression profiling to study both the molecular biology of cancer and the cellular physiology of difficult-to-isolate cell types has led to a growing need for methods that allow the use of limiting quantities of RNA. Small surgical biopsies, fine needle aspirates, cyto-lavages, punch biopsies and blood samples often yield only 1–4 μg quantities of RNA as starting material for expression profiling. This limitation has prompted the development of amplification methods that produce the quantities of RNA required for microarray analysis. Changing requirements for the type and quantity of amplified RNA, driven by evolving microarray technologies, have led to the development of novel amplification strategies. While current methods are capable of delivering high-yield RNA amplification, this is often only achieved after complex priming strategies (for example, involving 4 or more primers) are coupled with multiple rounds of PCR and/or + sequence on the second-strand cDNA could then be used to facilitate amplification. These methods were confounded by differential amplification of cDNA and by introduction of errors by Taq polymerase. This problem prompted the development of a linear, T7-based in vitro transcription (IVT) method by Van Gelder and Eberwine [Early attempts to amplify RNA employed a strategy based upon the Polymerase Chain Reaction (PCR) -4. TheseEberwine -7.E. coli RNAse H, and priming for second-strand cDNA synthesis occurs by either RNA nicking and priming or cDNA hairpinning6. Second-strand cDNA synthesis is carried out with E. coli DNA polymerase and E. coli DNA ligase followed by blunt-ending with T4 DNA polymerase. Transcription and amplification are then accomplished using the T7 RNA polymerase, which binds to the T7 promoter introduced during first-strand cDNA synthesis, producing antisense RNA (aRNA).In what has now become known as the "Eberwine Method," RNA templates are primed with an oligo(dT) primer that has been 5' modified to contain a promoter for the T7 RNA polymerase and are subsequently reverse transcribed into first-strand cDNA. The RNA-cDNA hybrid is then treated with Technical revisions of the Eberwine method have included changes in first-strand primer concentration to minimize the appearance of non-sequence dependent RNA in the amplified product , supplemOne such alternative  is basedA second alternative to the Eberwine method is the "template-switching" (TS) strategy . This teA third recently introduced alternative is Ginsberg's Terminal Continuation (TC) technique . In this2(ratio) differences within ± 0.5 of the unamplified RNA, they concluded that RNA amplification is useful in expression profiling and is likely to assist in the measurement of low-abundance RNA.The degree to which the pool of amplified RNA generated by these methods reflects the unamplified sample from which it is derived is an obvious concern for microarray applications and other downstream analyses, and the history of attempts to validate RNA amplification methods is summarized by Nygaard et al. . BrieflyDespite the methodological validation provided by these studies, only recently have statistically-based analyses been used to compare RNA amplification methods in a critical, head-to-head fashion. A study by Zhao  examined+ RNA [It is currently accepted that RNA amplification strategies based on the Eberwine method maintain relative mRNA levels between samples when amplifying from either total RNA or poly(A)+ RNA ,25, are + RNA , and are+ RNA . AccordiFirst, most commercial protocols produce RNA only in the antisense orientation. With the emergence of spotted oligonucleotide arrays and mixed cDNA/oligo arrays , appropr+ RNA extracted from both ideal samples (e.g. cell line RNA) and "real-world" samples (e.g. tumors or tissues). This protocol should avoid the need for PCR steps and should require a minimum number of primers (two). Additionally, the protocol should be cost-effective, efficient, and technically simple to conduct. Finally, the method should give results consistent with similar amplification techniques when used with subsequent microarray analysis. We believe these criteria have been met with our protocol, which consequently will be useful in a variety of laboratory applications.Our goal was to develop a strategy based upon the Eberwine method but with the ability to produce sense RNA from small quantities of total or poly-(A)Quantitation of first strand cDNA after hydrolysis of the cDNA/RNA hybrid shows that priming with the T7-Oligo(dT) promoter/primer followed by reverse transcription produces cDNA that totals 5–10% by mass of the original mass of total RNA (data not shown). This is consistent with the average percentage of mRNA in the total RNA sample and therefore suggests that priming proceeds efficiently and that first strand cDNA synthesis successfully copies the population of mRNA in the original total RNA sample. Quantitation of second strand cDNA after priming with the T3N9 promoter/primer indicates a 120% increase in mass versus the mass of the first strand cDNA (data not shown). This suggests that second strand priming proceeds efficiently and that the first strand cDNA is successfully converted to double stranded cDNA by the second strand synthesis reaction. The extra mass (in excess of 100%) is attributable to the use of random nonamer primers which initiates synthesis at multiple positions along each first-strand cDNA template.While inherent constraints of reverse transcription limit the amount of truly "full-length transcript" produced during any RNA amplification scheme, it is nonetheless desirable for an amplification method to favor the production of long transcript. RNA amplified using our method has a median length of 794 nucleotides (range: 70–9000 nt) suggesting that this method is capable of preserving long transcript during amplification Table .) of 26.3 μg of amplified sense mRNA from 4 μg of total RNA (Table Our method produced an average (2(ratio) plots of fluorescence intensity in amplified versus unamplified samples were constructed and analyzed for correlation as described above. Our results show that the amplification bias introduced by this method is small, as reflected in the high average correlation coefficient  between expression profiles from amplified and unamplified RNA samples ( = 0.8009, Table Any amplification will introduce some degree of bias into the population of amplified RNA. While recent reports suggest that the magnitude of this bias may be relatively unimportant as long as the bias is highly reproducible , we feel2(ratio) plots of fluorescence intensity were constructed and analyzed as described above. Our results demonstrate a high degree of correlation between independent replicates , suggesting that the amplification is highly reproducible and that the amplification bias is introduced consistently when the protocol is repeated.We evaluated the reproducibility of this method and the consistency of the propagation of the amplification bias by comparing the expression profiles of hybridizations of three independently amplified RNA samples. LogWe compared these results to results from the RiboAmp mRNA amplification kit (Arcturus), which produces antisense RNA using a version of the Eberwine method, and from the BDSmart mRNA amplification kit (BD Biosciences), which produces sense RNA using a template-switching strategy. Both protocols were carried out according to the manufacturer's specifications using 4 μg of the pooled cell line RNA and the glioblastoma RNA as the starting material for amplification. The final yield of each method was measured using the NanoDrop spectrophotometer, the median size of the amplified mRNA from each kit was assayed using the Bioanalyzer 2100 (Agilent), and the amplification bias of each method was assessed using microarray analysis (as described above).) and the dispersion of each group (σ2, CV) were calculated, demonstrating a high degree of reproducibility for both the RiboAmp ( = 0.950, σ2 = 0.0004, CV = 2.09%) and the BDSmart ( = 0.895, σ2 = 0.0010, CV = 3.47%) protocols. These results suggest that subsequent comparisons of our amplification method to these commercial techniques are not confounded by individual variability among replicates. Additionally, the strong correlation between independent replicates of our method (see above) and of these commercial techniques suggests that performing additional independent replicates would not add significant statistical power to subsequent comparisons.Three independent replicates were conducted for each method, and the reproducibility of the amplification was assessed as described above. The mean correlation coefficient ( = 23.6 μg) was less than that of the Arcturus RiboAmp method ( = 71.6 μg, p < 0.04) but greater than that of the BDSmart method ( = 4.87 μg, p = 1.7 × 10-14). The decreased yield versus the Arcturus RiboAmp method is expected, because IVT with the T7 promoter (production of antisense RNA, Arcturus method) proceeds more efficiently than IVT with the T3 promoter (production of sense RNA, our method). The improved yield over the BDSmart method is notable considering that both of these methods involve production of amplified sense RNA (Table The average yield from our method  = 3.6 μg wa = 0.8009) versus amplification with the Arcturus RiboAmp method ( = 0.7202, p < 0.01) and was comparable to the correlation after amplification with the BDSmart method ( = 0.7679, p = 0.15) (Table The median length of the amplified RNA from our method (794 nt) was greater than that of the Arcturus RiboAmp method (507 nt, p < 0.003) and comparable to that of the BDSmart amplification method (764 nt, p = 0.88) Table . The corIn vitro Transcription5–6 became the first standard protocol for RNA amplification designed to fulfill these requirements. The evolution of microarray technology and other RNA techniques have imposed requirements on the type, length, and/or orientation of the starting RNA. Additionally, the growing interest in medical and microbial genomics now requires that data be collected from samples that are becoming progressively smaller and more difficult to acquire. This changing role for RNA amplification has catalyzed the development of multiple institutional and commercial amplification protocols, all of which claim to be high-yield, low-bias techniques capable of amplifying RNA with the specific characteristics required for downstream applications.The emergence of methods to study the transcriptome initially necessitated the production of relatively large quantities of RNA from experimental systems, and Eberwine and Van Gelder's T7-based system of Template switching, terminal continuation, and other novel techniques for RNA amplification that have been developed over the past five years have become increasingly technically complex, and we feel that this is a significant disadvantage. In particular, they stray from the Eberwine approach, which has been extensively validated, and often rely on the use of proprietary (i.e. unspecified) enzymes, primers, and reaction components, which limit the utility of the methods (as well as protocol modification and fine-tuning). While proponents of these methods argue that the limitations are offset by improved yield, production of long transcript, reproducibility, and reduced amplification bias, we have found in practice that no single protocol is able to perform consistently in all four areas.The technique that we have described affords the advantages of all of the aforementioned protocols while eliminating the major limitations and controlling the technical complexity. Several aspects of this protocol make it a robust tool that we believe will be useful in conjunction with a variety of experimental systems and downstream applications. First, the method we present is based upon the Eberwine technique. The methods are both straightforward and validated and do not require the use of custom enzymes, multiple (more than 2) proprietary primers, or PCR steps to complete the amplification. Second, this new method produces amplified mRNA in the sense orientation and can be used for production of aRNA with minor modification. This corrects for the downstream limitations imposed by antisense amplification in both the original Eberwine method and the Arcturus RiboAmp kit and allows amplified mRNA produced by our method to be used in conjunction with both cDNA (sense and antisense orientation) and long oligonucleotide (sense orientation) arrays. Third, our method requires only one round of amplification to produce RNA in the sense orientation. We believe that this represents a significant advantage over previously described, T7-based amplification strategies (most notably the method of Xiang ), where Another advantage of our protocol is that it is readily adaptable to a variety of experimental systems. The approach that we have described synthesizes amplified RNA from eukaryotic total RNA in the sense orientation. However, because this method adds unique promoters to both the first and second cDNA strand (T7 and T3, respectively), either orientation of RNA can be produced from the same population of cDNA simply by selecting the appropriate RNA polymerase for IVT. Moreover, changing the two primers allows the protocol to be adapted to produce sense or antisense RNA from either prokaryotic or eukaryotic total RNA without any additional adjustments. The protocol can also be modified for use with either T3 or T7 IVT systems by changing the promoter sequence of the promoter-modified primer, it can be used to salvage partially-degraded RNA (as described by Xiang ), and itIn clinical and research settings using laser capture microdissection or other strategies where very small amounts of starting RNA (10–100 ng) are available, multiple rounds of amplification may still be necessary. A final advantage of our protocol, as discussed previously, is that a second round of amplification can be easily incorporated and can be used to produce additional RNA in either the sense or antisense orientation. Future testing of this protocol will focus on the quantity and fidelity of the RNA population after multiple rounds of amplification and will include appropriate comparisons to commercial, multi-round techniques. However, the purpose of the present technique is to minimize bias while maintaining yield after a single round of amplification. In this setting, our results show that our method outperforms two commercial, single-step protocols when using identical quantities of initial RNA. We believe the performance and versatility of this method make it particularly beneficial to investigators attempting to conduct microarray analysis from 1–4 μg of initial RNA.Neither this nor any other current strategy for RNA amplification is optimally suited to every experimental setting, and each technique has its own advantages and limitations. The challenge of selecting an amplification strategy is choosing a method that provides sufficient amplification and appropriate RNA orientation for downstream requirements yet minimizes the labor, cost, and bias introduced by the process. The method that we have described has been designed and validated for use in experiments where microarray analysis is to be performed with approximately 1 μg of starting total RNA. In this setting, our method produces a population of amplified RNA having minimal and consistent amplification bias, orientation compatible with both spotted cDNA and oligonucleotide arrays, and quantity sufficient for several downstream assays from a single amplification. Additionally, the protocol we present here can be easily adapted for use in a variety of experimental systems. This includes production of either sense or antisense RNA in prokaryotic or eukaryotic systems (simply changing one or both primers). None of these variations require multiple rounds of amplification to produce RNA of a specific orientation, but the protocol can easily be modified to enable multiple rounds when necessary.Finally, we would like to note that there are some limitations to the method we have outlined here. Readers may note that several amplification strategies have been reported to produce greater than 1,000-fold increases in RNA yield, one aspect of our method that may be viewed as a limitation is its ~130-fold amplification. However, most higher-yield amplification strategies use multiple rounds of amplification and are subject to additional amplification bias. While we recognize that researchers using submicrogram quantities of starting material may consider strategies incorporating multiple rounds of amplification, in our experience even small tissue biopsies yield more than 1 μg of total RNA. The ability to amplify this quantity of RNA efficiently and effectively was one of our major design considerations. In this setting, 130-fold amplification produces more than enough RNA for multiple downstream microarray analyses and confers the benefit of reduced bias inherent in a single-round amplification protocol.We have developed a robust method for amplification of mRNA in the sense orientation. This protocol is based upon the Eberwine method and incorporates the advantages of more recent amplification techniques while eliminating many of the limitations of these strategies. Our method allows the production of sense mRNA with one round of IVT, yields 130-fold amplification, preserves long transcript, and produces mRNA that is well suited for downstream microarray applications. Microarray assays performed with RNA amplified using our protocol demonstrate that the method results in low amplification bias and is highly reproducible. Additionally, our method is readily adaptable for the production of sense or antisense, labeled or unlabeled RNA from intact or partially-degraded samples of prokaryotic or eukaryotic total RNA. The method outperforms several commercial RNA amplification kits and can be used in conjunction with a variety of downstream microarray platforms (cDNA arrays, oligonucleotide arrays, Affymetrix GeneChip™ assays). We feel that these advantages make our method a robust tool with the potential for application in a variety of research settings.24 primer followed by heat denaturing and then cooling to facilitate primer annealing. Reverse transcription produces cDNA-mRNA hybrids which are then subjected to alkali hydrolysis to remove template mRNA. First-strand cDNA (fs-cDNA) is separated from residual enzymes, nucleotides, and mRNA fragments using a spin column technique.The protocol that we have designed is based on the Eberwine method but uses novel priming strategies to produce amplified RNA in the sense orientation. First-strand cDNA synthesis from total RNA is primed with an Oligo(dT)E. coli DNA polymerase and ligase followed by blunt-ending with T4 DNA Polymerase. The double-stranded cDNA is purified using a spin column technique, and in vitro transcription (IVT) from the T3 promoter sequences incorporated into the ss-cDNA produces amplified RNA in the sense orientation . The sample is heated to denature the fs-cDNA and to eliminate secondary structure. The temperature is rapidly dropped to the upper limit of the annealing range and then ramped more slowly to a final temperature of 4°C. We believe that this strategy minimizes "self-priming" of the fs-cDNA and, in the absence of competitive priming from RNA fragments, facilitates optimal annealing of the second-strand primer. Second-strand cDNA (ss-cDNA) synthesis is carried out using n Figure .2 and maintained accordingly. When culture plates achieved ~90% confluence, RNA was extracted and purified following TIGR standard operating procedures based on the Trizol method (Invitrogen) [260/280 and OD230/260 in TE (pH 8.0) using a NanoDrop spectrophotometer, and RNA integrity was assessed by measurement of the 28S/18S ratio with the Agilent Bioanalyzer 2100 using the Agilent Total RNA Nano chip assay. The final values for the RNA pool were: OD260/280 = 2.04, OD230/260 = 2.20, 28S/18S = 2.13, indicating high purity, non-degraded RNA. Superas-In RNAse inhibitor (Ambion) was added to a final concentration of 1U/μL according to manufacturer's recommendations. RNA was stored at -80°C until it was used in the RNA amplification process.Total RNA used in this study was derived from three cell lines: PA-1 ovarian teratocarcinoma, CaOV-3 ovarian adenocarcinoma, and U118MG glioblastoma. All cell lines were grown in a monolayer under Dulbecco's minimum essential medium (DMEM) with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Cell lines were held at 37°C in an atmosphere of 4% COitrogen) . RNA extAll temperature controlled steps in this protocol were conducted using a Peltier thermal cycler (PTC225 DNA Engine Tetrad, MJ research) in thin-walled, nuclease-free PCR tubes (BioRad). All enzymes, buffers, and other reaction components were purchased from Invitrogen unless otherwise noted. Four (4) μg of pooled total cellular RNA (described above) was as starting material for the amplifications. Priming for first strand cDNA synthesis is carried out by combining the total RNA with 1 μL of T7-Oligo(dT) primers (100 ng/μL, Ambion) and 1 μL of Superas-In RNAse inhibitor (20U/μL, Ambion). The mixture is diluted to 17.8 μL in DEPC treated water, heated at 70°C for 10 minutes, and cooled to 4°C for five minutes. First strand cDNA synthesis is accomplished by adding 6 μL of 5× First Strand Buffer, 3 μL 0.1M DTT, 1.2 μL of dNTP mix (50 mM), and 2 μL of SuperScript II reverse transcriptase followed by incubation at 42°C for 2.5 hours. After incubation, the reaction is cooled to 4°C for 2 minutes. Template RNA is hydrolyzed by adding 10 μL of 1N NaOH (Sigma) and 10 μL of 0.5M EDTA (Sigma) followed by incubation at 65°C for 15 minutes. The pH is subsequently neutralized by adding 10 μL of 1N HCl (Sigma). Hydrolyzed RNA and residual dNTPs are removed using the Minelute reaction cleanup kit (Qiagen) according to the manufacturer's protocol. Two rounds of elution from the column, each in 10 μL of Buffer EB (Qiagen) are performed to improve recovery.11 (Operon) followed by incubation at 95°C for 3 minutes. Immediately following this incubation, the samples temperature is decreased as quickly as possible to 50°C followed by a temperature ramp from 50°C to 4°C at the rate of 0.4°C/s (~120s ramp time) and then a hold at 4°C for 2 minutes. Second strand cDNA synthesis is accomplished by adding 7μL of 5× Second Strand Buffer, 2 μL of dNTP mix (50 mM), 4 μL of E. coli DNA polymerase I (10U/μL), and 1 μL of E. coli DNA ligase (10U/μL) followed by incubation at 16°C for 2 hours. After incubation, blunt-ending is carried out by adding 2 μL of T4 DNA polymerase (5U/μL) and incubating at 16°C for 5 minutes. The reaction is terminated by adding 3.5 μL of 0.5 M EDTA (Sigma). Double stranded cDNA is purified using the Minelute reaction cleanup kit (Qiagen) following the manufacturer's protocol. Two rounds of elution with Buffer EB (Qiagen) are carried out to improve yield. The first elution is in 10 μL of Buffer EB, and the second is in 7 μL of Buffer EB. This provides the eluted cDNA in a final volume of 16 μL (corrected for 1 μL of retention on the column), which is the appropriate volume for use in the subsequent IVT reaction.Priming for second strand cDNA synthesis is accomplished by combining the eluted first-strand cDNA with 2 μL of random nonamer primers modified by the addition of a T3 promoter sequence at the 5' end (T3N9 primer, 100 ng/μL, Table In vitro transcription is achieved using the T3 MegaScript kit (Ambion). A double reaction is used to improve yield, so 16 μL of NTP mix, 4 μL of 10× Reaction Buffer, and 4 μL of T3 MegaScript Enzyme Mix are added to the sample. The reaction is incubated at 37°C for 14 hours (overnight). The amplified RNA is purified using either the RNEasy Mini or the RNEasy Minelute kit (Qiagen) according to the manufacturer's protocol. Superas-In RNAse inhibitor (Ambion) is added to the amplified RNA to a final concentration of 1U/μL according to the manufacturer's recommendations.260/280 and OD230/260 in the corresponding elution buffers (pH 8.0) using a NanoDrop spectrophotometer. RNA length distribution and integrity were assessed with the Agilent Bioanalyzer 2100 using the Agilent Total RNA Nano chip assay. Agilent's Bioanalyzer Expert 2100 software was used to quantify transcript length, and the software's automatic integration tool was used to determine the area under the curve. The median transcript length for each method was taken as the point at which half of the integrated area was achieved.cDNA and RNA quantity and purity were assessed by measurement of OD28. cDNA target synthesized from the cell line RNA was indirectly labeled with Cy-5 while target derived from the glioblastoma was labeled with Cy-3. The labeled cDNA from the cell line pool and from the glioblastoma were cohybridized to human 32,000 element spotted cDNA arrays. The array production, cDNA synthesis, target labeling, and hybridization were conducted following TIGR standard operating procedures [Microarray analysis was conducted to determine the magnitude of amplification bias. Pooled human cell line RNA (as described above) as well as RNA extracted from one human glioblastoma were amplified as described. The amplified sense RNA (2 μg) was used as the starting material for synthesis of cDNA target, and a spiking control consisting of RNA transcripts of 10 genes from the Arabidopsis thaliana genome was added to each sample prior to cDNA synthesis in order to provide a consistent positive reference in subsequent hybridizationsocedures . IndepenIn order to compare our method to commercially-available RNA amplification kits, microarray analysis was conducted using identical pooled cell line and glioblastoma RNA that was amplified with two commercial products . Amplification of 4 μg of total RNA was carried out according to the manufacturer's protocol for both kits, and 2μg of amplified RNA from each sample was used for microarray analysis as described above. Independent replicates were conducted for all amplifications and hybridizations.Unamplified total RNA from both the pooled cell line and the glioblastoma was used as the starting material for the control arrays. Ten (10) μg of total RNA from each sample was used for cDNA synthesis, and the remainder of the protocol was carried out as described for the amplified samples.2 transformed, and  was calculated for each array element. Assessments of bias were conducted by comparing the log2(ratio) values for each element in the amplified sample arrays to their counterparts in the unamplified control arrays. Measurements of reproducibility were conducted by comparing the log2(ratio) values of corresponding elements in independent replicates.Microarray data was analyzed using the TIGR TM4 software package for microarray analysis . TIGR Sp against the  and calculating the correlation coefficient (r) for each independent replicate. The mean correlation coefficient  was calculated for each method, and differences between  for each amplification method were tested for statistical significance. Welch's T-Test [The degree of bias introduced by an amplification method was assessed by plotting s T-Test  was used2(ratio) values for paired hybridizations derived from independent amplifications of the same starting RNA, calculating the correlation coefficient (r) for a series of independent replicates, and computing the mean correlation coefficient . Reproducibility was further investigated by calculating the variance (σ2) and the coefficient of variation  for  (CV), which describe the dispersion of the values contributing to .The degree of reproducibility of our amplification method was assessed by plotting the logThis method is based on an idea by Norman Lee. John Quackenbush and Nicholas Marko further developed the approach. Marko reduced this to practice in the laboratory, greatly expanding on the method and creating a robust protocol. Bryan Frank contributed significantly to the development, implementation, and optimization of the protocol. Marko conducted the data analysis and was largely responsible for the present manuscript.


The phosphatidylserine receptor (Ptdsr) on phagocytes has been implicated in the recognition and engulfment of apoptotic cells and in anti-inflammatory signaling. To determine the biological function of the phosphatidylserine receptor Ptdsr function in mice causes perinatal lethality, growth retardation and a delay in terminal differentiation of the kidney, intestine, liver and lungs during embryogenesis. Moreover, eye development can be severely disturbed, ranging from defects in retinal differentiation to complete unilateral or bilateral absence of eyes. Ptdsr -/- mice with anophthalmia develop novel lesions, with induction of ectopic retinal-pigmented epithelium in nasal cavities. A comprehensive investigation of apoptotic cell clearance in vivo and in vitro demonstrated that engulfment of apoptotic cells was normal in Ptdsr knockout mice, but Ptdsr-deficient macrophages were impaired in pro- and anti-inflammatory cytokine signaling after stimulation with apoptotic cells or with lipopolysaccharide.Ablation of Ptdsr is essential for the development and differentiation of multiple organs during embryogenesis but not for apoptotic cell removal. Ptdsr may thus have a novel, unexpected developmental function as an important differentiation-promoting gene. Moreover, Ptdsr is not required for apoptotic cell clearance by macrophages but seems to be necessary for the regulation of macrophage cytokine responses. These results clearly contradict the current view that the phosphatidylserine receptor primarily functions in apoptotic cell clearance. Programmed cell death, or apoptosis, is required for the normal development of almost all multicellular organisms and is a physiological mechanism for controlling cell number; as a result, structures that are no longer needed are deleted during development and abnormal cells are eliminated ,2. Most Drosophila and Caenorhabditis elegans have recently yielded evidence that basic phagocytic mechanisms and pathways for the recognition and engulfment of apoptotic cells are highly conserved throughout phylogeny [vβ3, and members of the collectin family and their receptors CD91 and calreticulin [in vivo is supported by the observation that autoimmune responses can be provoked in mice when key molecules for apoptotic cell recognition and uptake are missing. This has been reported for knockout mice lacking the complement protein C1q [Mer [Almost all cell types can recognize, respond to, and ingest apoptotic cells by using specific sets of phagocytic receptors that bind to specific ligands on apoptotic cells. Detailed genetic studies in hylogeny ,8. In veeticulin . The indeticulin ,6,14. Thtein C1q , for micC1q [Mer  and, morC1q [Mer ,18.vβ3 [The exposure of the phospholipid phosphatidylserine (PS) in the outer leaflet of the plasma membrane of apoptotic cells has been described as one of the hallmarks of the induction of apoptosis and is considered to be one of the most important signals required for apoptotic cell recognition and removal . A numbevβ3 ,24. Othevβ3  and refeIn vitro, it has been shown that the Ptdsr can mediate the uptake of apoptotic cells and that such Ptdsr-mediated phagocytosis can be inhibited through addition of PS liposomes, the PS-binding molecule annexin V or an anti-Ptdsr antibody [The best-characterized molecule so far that binds PS in a stereo-specific manner is the phosphatidylserine receptor (Ptdsr) . In vitrantibody . Moreoveantibody ,27. Thesantibody .Hydra showed an exclusively nuclear localization for the Ptdsr protein [Hydra epithelial cells did not change upon phagocytosis of apoptotic cells. These reports challenge the original hypothesis, according to which Ptdsr is an exclusively transmembrane receptor for apoptotic cell recognition and anti-inflammatory signaling.Very recently, Ptdsr has been found in the cell nucleus. Its nuclear localization is mediated by five independent nuclear localization signals, each of which alone is capable of targeting Ptdsr to the cell nucleus . Moreove protein . Most inin vivo, we performed gene-expression and gene-targeting studies in mice. A perinatally lethal phenotype was observed in Ptdsr-knockout mice, and Ptdsr-deficient embryos displayed multiple defects in tissue and organ differentiation. While this work was in progress, both Li et al. [et al. [Ptdsr-knockout mice. Of note, although some of their results were confirmed in our study, we found a fundamentally different phenotype with regard to clearance of apoptotic cells. Moreover, our study revealed marked and unexpected findings in Ptdsr-deficient mice that are not related to apoptosis.To examine further the role of Ptdsr i et al.  and Kuni [et al.  also repin vivo the functions of the phosphatidylserine receptor Ptdsr, we generated a null allele in the mouse by gene targeting . Heterozygous Ptdsr+/- mice were viable and fertile and showed no obvious abnormalities. Ptdsr +/- mice were intercrossed to generate homozygous Ptdsr-deficient mice. The absence of Ptdsr expression in Ptdsr -/- embryos was confirmed by RT-PCR (data not shown), and by northern and western blotting analyses  9.5 and E18.5, 198 (19.2%) Ptdsr-deficient homozygous embryos were harvested, indicating that the introduced mutation is associated with a low rate of embryonic lethality in utero.To investigate g Figure . In contout mice ,32, we uout mice , thus ges Figure . Interbrg Figure  of embryPtdsr -/-  embryos were viable and of normal size. At E13.5 and thereafter, however, most Ptdsr -/-embryos showed morphological abnormalities  showed that Ptdsr is expressed early in development, because we were able to detect Ptdsr transcripts in ES cells and embryos at all developmental stages. To analyze in more detail the temporal and spatial expression patterns of Ptdsr, and to correlate expression patterns with observed pathological malformations, we made use of a Ptdsr-β-geo gene-trap reporter mouse line generated from a Ptdsr gene-trap ES cell clone. This line has an insertion of β-galactosidase in the 3' region of the gene .We first examined s Figure . At E10.s Figure . Ptdsr en Figure . Transven Figure . Expresss Figure . We carePtdsr is ubiquitously expressed throughout embryogenesis and in adult tissues, although at different levels.Hybridization of a multiple-tissue northern blot revealed a single transcript of about 1.8 kb in almost every tissue analyzed in adult mice Figure . The mosPtdsr in organ development. Serial histological sections of Ptdsr -/- and control embryos were taken to perform a detailed morphological analysis of all organ systems during development. A significant delay in organ and tissue differentiation was observed at E16.5 in lungs, kidneys and intestine. Lungs of control littermates were properly developed with expanding alveoli  also displayed extensive brain malformations that resulted in externally visible head abnormalities, with occasional ectopic tissue outside the skull or exencephaly  in mildly affected homozygotes to anophthalmia in severely affected Ptdsr -/- mice that was associated with induction of ectopic eye structures in nasal cavities.We next examined s Figure  by serias Figure . Most sum Figure . Examinam Figure . This stPtdsr is functionally required for the clearance of apoptotic cells. We started with an investigation of cell death in vivo in the interdigital areas of the developing limbs. Apoptosis of interdigital cells in the distal mesenchyme of limb buds occurs most prominently from developmental stages E12.0 to E13.5 and can be easily examined in situ by whole-mount terminal deoxynucleotide transferase-mediated UTP end-labeling (TUNEL). We compared the pattern of interdigital cell death in fore and hind limb buds from Ptdsr -/- (n = 3) and Ptdsr +/+ (n = 3) mice at E12.5 and E13.5. No differences in accumulation of TUNEL-positive cell corpses were observed between the two genotypes  and analyzed additional organs and tissues where apoptosis plays a crucial role in tissue remodeling during development. Starting at E12.5, we analyzed and compared the number and distribution of aCasp3-positive cells in over 140 serial sections of three wild-type and six Ptdsr -/- embryos in consecutive and corresponding sections. The sagittal sections were separated by 5 μm, allowing a detailed analysis of apoptosis in several organs and tissues. Tissue restructuring by programmed cell death occurred most notably within the ventral part of the neural tube  and quantified their phagocytosis rates. Phagocytosis of apoptotic thymocytes was investigated at 60, 90 and 120 minutes after addition of target cells in the absence of serum. Analysis of phagocytosis rates by flow cytometric analysis (FACS) revealed no differences in the efficacy of apoptotic cell uptake between Ptdsr -/- and Ptdsr+/+ macrophages and demonstrated no differences in apoptotic cell engulfment between selected time points (data not shown). To re-examine and further independently validate the result of normal apoptotic cell uptake by Ptdsr -/- macrophages, we performed phagocytosis assays for 60 min and determined the percentage of macrophages that had engulfed apoptotic cells, in a total of at least 300 macrophages counted by fluorescence microscopy. Phagocytosed, 5-carboxytetramethylrhodamine- (TAMRA-) labeled apoptotic cells were identified as being engulfed by inclusion in F4/80-labeled macrophages. Analysis was done independently by three investigators who were not aware of macrophage genotypes (Ptdsr -/- or Ptdsr+/+). Again, no differences were found in the percentage of macrophages that had engulfed apoptotic cells  after stimulation of FLDMs with lipopolysaccharide (LPS), with and without co-culture of apoptotic cells. Quantification of TGF-β1 and IL-10 levels after 22 hours of culture demonstrated that Ptdsr -/- macrophages were able to secrete these anti-inflammatory cytokines upon ingestion of apoptotic cells, although at a slightly lower level than wild-type  at different time points after stimulation . These results indicate that Ptdsr is not required in macrophages for the inhibition of pro-inflammatory signaling after recognition and engulfment of apoptotic cells. Ptdsr-deficiency does, however, affect the overall release of pro- and anti-inflammatory cytokines after stimulation with LPS and after double treatment with LPS and apoptotic cells, indicating that Ptdsr-deficient macrophages have a reduced capacity to produce or secrete pro- and anti-inflammatory cytokines.To analyze whether pro-inflammatory signaling is affected in n Figure . Ptdsr -s Figure . Moreovephosphatidylserine receptor (Ptdsr) gene in C57BL/6J mice. We show that ablation of Ptdsr results in profound differentiation defects in multiple organs and tissues during embryogenesis, although with variable penetrance. While this work was in progress, two other groups reported the generation of Ptdsr-deficient mice [Ptdsr-knockout mouse lines differ in the genetic background in which the mutation was generated and maintained, however. In our case, the Ptdsr-null allele was generated in an isogenic C57BL/6J background, whereas Li et al. [et al. [Ptdsr-knockout mice in a mixed 129 × C57BL/6 background. The ablation of Ptdsr function results in perinatal lethality in all cases, but there are interesting differences in severity or expressivities of phenotypes among the different Ptdsr-deficient mouse lines. This might be due either to differences in genetic background or because the phenotypes that have been investigated in this study have not been analyzed in such detail before.In this study, we have generated a null mutation in the ent mice ,32. In aent mice  and thisi et al.  and KuniPtdsr-knockout mouse line reported here, growth retardation started from E12.5 onwards and was associated with delayed differentiation in several organs in which Ptdsr is expressed either during embryogenesis or later in adulthood. At E16.5 almost no branching morphogenesis of the lung epithelium was observed in Ptdsr -/- lungs. Similarly, epithelial structures were only partially developed in mutant kidneys, without terminal differentiation of Bowman's capsule and with a severe reduction in the number of differentiated collecting tubules. Likewise, the differentiation of the intestine was also severely delayed at this developmental stage. When compared with wild-type controls, intestinal tissues of Ptdsr knockout mice appeared unstructured, with an absence of enteric ganglia and of differentiated smooth muscle tissue. Interestingly, defects in kidney and intestine differentiation were not described in the Ptdsr-knockouts generated by Li et al. [et al. [Ptdsr-/- embryos shortly before birth (E18.5) or neonatally, we found only mild differentiation delays in organs that appeared severely affected at mid-gestation. This 'recovery' was most visible in Ptdsr -/- lungs: at P0 we found expanded lungs in the knockout mice that showed normal branching patterns, with differentiated alveoli and bronchioles.In the i et al.  and Kuni [et al. . SurprisPtdsr-knockout mice throughout embryogenesis (E16.5 to P0). Comparative immunohistochemistry for aCasp3 revealed that apoptosis is a rare event during lung morphogenesis. Furthermore, we failed to detect any differences in the number of apoptotic cells in Ptdsr-knockout and wild-type animals in the rare cases where we could detect apoptotic cells within lung tissues. These findings are contrary to the results reported by Li et al. [Ptdsr-deficient mice. In contrast, our findings are in line with the current view on lung development during embryogenesis. Accordingly, formation of the epithelial lung via branching morphogenesis can be subdivided into a series of sequential steps that involve: first, formation of the organ anlage in the form of a placode; second, primary bud formation by placode invagination; third, branch initiation and branch outgrowth; fourth, further reiteration of the branching process; and fifth, terminal differentiation of organ-specific proximal and distal structures [Ptdsr -/-neonates were almost fully expanded and appeared normal in structure in comparison to wild-type littermates, it is highly unlikely that Ptdsr mutants die of respiratory lung failure. In addition, Li and colleagues [Ptdsr-deficient animals, supporting the idea of normal maturation of surfactant-producing type II alveolar epithelial cells and lung function. Other defects must therefore be responsible for the death of Ptdsr-mutant mice. The frequently observed subcutaneous edema of various extents in Ptdsr-deficient homozygotes gave us a hint that Ptdsr-deficiency and lethality might be associated with cardiovascular problems. Indeed, very recently we have obtained strong evidence that Ptdsr-knockout mice die as a result of defects in heart development that are associated with specific cardiopulmonary malformations; (J.E. Schneider, J.B., S.D. Bamfort, A.D.G., C. Broadbent, K. Clarke, S. Neubauer, A.L. and S. Battacharya, unpublished observations).We investigated the occurrence of programmed cell death during lung development in wild-type and i et al. , who sugructures ,35. In cructures ,36. Finalleagues  demonstrPtdsr gene. Ptdsr-deficient embryos can be roughly divided into two categories. The first, severely affected group develops anophthalmia that correlates with formation of ectopic retinal-pigmented epithelium and induction of proliferation of underlying mesenchyme in the nasal cavity. This phenotype represents a completely novel lesion that to our knowledge has not been described before in any other mouse mutant. The second group shows normal external eye structures, although in this case retinal development is temporally delayed during mid-gestation, with persistent, abnormal morphogenesis of the inner granular retinal layer at later stages of embryogenesis. A possible explanation for these two phenotypes can be found in the expression pattern of the Ptdsr gene. Initially, Ptdsr is expressed throughout the whole developing nervous system, with exceptionally high levels in the anterior part of the forebrain. Later expression becomes more restricted to the developing retina and lens. Thus, Ptdsr might play an important role in early events of ocular morphogenesis, such as establishment and bisection of eye fields and formation of optic cups. These early eye-formation steps are closely interconnected with development of the forebrain [In addition, we demonstrate that eye development requires a functional orebrain ,38 and torebrain -41.Ptdsr-knockout embryos that were associated with bilateral anophthalmia , but these do not resemble to any extent the brain-overgrowth phenotypes of caspase- or Apaf1-knockout mice . Moreover, in contrast to our studies, neither Li et al. [et al. [Ptdsr-deficient macrophages.In contrast to Li et al. , we did i et al.  found thi et al.  nor Kuni [et al.  determinPtdsr+/+ and Ptdsr -/- macrophages in the secretion of pro- and anti-inflammatory cytokines after stimulation with LPS and apoptotic cells. This provides evidence that cellular activation and effector mechanisms are impaired in Ptdsr-deleted macrophages. It remains to be determined which classical pathways of macrophage activation and function involve Ptdsr. This is especially important in light of recent findings that demonstrated nuclear localization of the Ptdsr protein [Interestingly, we observed differences between  protein .C. elegans are also contradictory. Wang et al. [psr-1, the C. elegans homolog of Ptdsr, is important for cell-corpse engulfment, whereas psr-1 RNAi studies performed by Arur et al. [psr-1 might act to transduce an engulfment signal upstream of Ced-2 (Crk II), Ced-5 (Dock 180), Ced-10 (Rac 1) and Ced-12 (Elmo) in one of the two cell-corpse engulfment pathways in the worm [psr-1 mutants and the complementation phenotypes in overexpressing transgenic worms shown by Wang et al. [C. elegans engulfment mutants [Most strikingly, the recently published data regarding the genetic ablation or perturbation of phosphatidylserine receptor function in g et al.  reportedr et al.  yielded,the worm . But theg et al.  are rath-5 Dock 10, Ced-10Ptdsr-deficient macrophages and fibroblasts . To our surprise, we observed similar staining patterns with cells of both genotypes. Furthermore, using a Ptdsr-peptide array we found that mAb 217G8E9 can bind weakly to a Ptdsr peptide, explaining the original isolation of Ptdsr cDNA clones by phage display [Many previous functional studies that reported a requirement for Ptdsr for the phagocytosis of apoptotic cells used the monoclonal anti-Ptdsr antibody mAb 217G8E9 . This an display ; but thePtdsr is essential for the differentiation and maturation of multiple tissues during embryogenesis. Ablation of Ptdsr function results in neonatal lethality and severe defects in the morphogenesis of several organs. The developmental malformations cannot be explained by impaired clearance of apoptotic cells, a process that proved to be normal in Ptdsr-deficient mice. This opens up the possibility either that there is an as-yet unknown Ptdsr receptor, which might act as a primary phosphatidylserine recognition receptor, or that recognition of phosphatidylserine and subsequent apoptotic cell engulfment and anti-inflammatory signaling are mainly mediated through phosphatidylserine bridging proteins and their cognate receptors. Although Ptdsr -/- macrophages were not impaired in their ability to phagocytose apoptotic cells, they showed reduced cytokine responses after stimulation. Further work will be required to determine the molecular mechanisms of these newly recognized Ptdsr functions during development.Our results demonstrate that Ptdsr-containing bacterial artificial chromosome (BAC) clone  was isolated by sequence homology from a C57BL/6J genomic BAC library . A 14.5 kb KpnI/BamHI fragment containing the entire Ptdsr locus and 5' and 3' flanking regions was subcloned from this BAC clone and a 1.9 kb RsrII/AatII fragment containing exons I and II of the Ptdsr gene was replaced by a 1.2 kb loxP-flanked neomycin-resistance gene cassette (neo).A KpnI-linearized targeting vector and selected with G418. ES-cell clones resistant to G418 were isolated and analyzed by Southern blot analysis for homologous recombination events within the Ptdsr locus. Chimeric mice were produced by microinjection of two independent homologous recombinant (Ptdsr+/-) ES cells into BALB/c blastocysts and transfer to pseudopregnant foster mothers followed by development to term. Chimeric males were mated with C57BL/6J females. From the two selected ES-cell clones, one successfully contributed to the germ-line. Germ-line transmission of the mutant allele was verified by PCR and Southern blot of genomic DNA from black coat-color F1 offspring.Bruce4 ES cells were transfected with β-geo gene-trap vector in the Ptdsr locus was identified by searching the BayGenomics database  with thPtdsr allele was detected using forward primer 1 (5'-GACACTGTCCATGGCAAACAC-3') and reverse primer 2 (5'-TAAAGTCGCCTTCCAGAAGATT-3'). The primer 1 site is located 5' to the deletion and the primer 2 site within the deletion. This primer pair amplified a fragment of approximately 300 bp from wild-type and Ptdsr+/- mice but not from Ptdsr -/-mutants. To detect the mutant Ptdsr allele, genomic DNA was also amplified using primer 1 and reverse primer 3 (5'-CCACACGCGTCACCTTAATA-3'), which corresponds to a sequence in the neo cassette. In this case, a 500 bp fragment was detected in mice heterozygous or homozygous for the mutant allele, while no signal was detected in wild-type mice. For Southern blot analysis, genomic DNA (30 μg) was digested overnight with BamHI  and ScaI , fractionated on a 0.8 % agarose gel, transferred to a nylon membrane  and hybridized with 5' and 3' flanking probes. The BamHI digest was hybridized with a Ptdsr-specific 5' flanking probe, and Southern blot gave a single 17.2 kb band for wild-type (+/+), an 11.6 kb band for homozygous (-/-) and both bands for heterozygous (+/-) mice. The ScaI digest was hybridized using a 3' flanking probe, and Southern blot gave a single 12.4 kb band for wild-type, a 17.2 kb band for homozygous and both bands for heterozygous mice.The genotypes of embryos or animals were determined by PCR analysis and confirmed by Southern blot. Genomic DNA for PCR was prepared from extraembryonic membranes or tail clips using a non-organic tail-DNA extraction protocol . High mo+ RNA northern blot (OriGene Technologies Inc., Rockville, USA) was hybridized using as the probe a Ptdsr fragment amplified from wild-type cDNA using forward primer 5'-GTTCCAGCTCGTCAGACTCG-3' and reverse primer 5'-TGCCCCTAAGACATGACCAC-3'. In all experiments the same membrane was re-hybridized with a β-actin probe (OriGene) to confirm that equivalent RNA levels were present in each lane. Northern blotting indicated that homozygous mutant embryos did not express Ptdsr mRNA and heterozygous mutant embryos expressed only reduced amounts of Ptdsr mRNA.Total RNA was isolated from homogenized embryos using TRIZOL reagent (Invitrogen GmbH, Karlsruhe, Germany). For northern blots, either total RNA (30 μg) was extracted from embryos, electrophoresed and transferred to a nylon membrane  or a polyAEmbryos (E13.5) for protein isolation were homogenized in lysis buffer containing 1 × PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS and protease inhibitor cocktail . Equal amounts (25 μg) of protein lysate were separated by SDS-polyacrylamide gel electrophoresis and transferred onto a PVDF membrane (Millipore, Billerica, USA) according to standard protocols. Western blots were done using a specific antibody to Ptdsr  and β-actin  as described by the supplier. Secondary antibodies conjugated to horseradish peroxidase were from Santa Cruz and Abcam, used as described by the supplier, and detection was performed with an enhanced chemiluminescence system .Ptdsrtm1Gbf mutant was crossed to C57BL/6J mice to establish the co-isogenic C57BL/6J-Ptdsrtm1Gbf mouse line. All studies were approved by the appropriate authorities.Wild-type C57BL/6J and 129P2/OlaHsd mice were obtained from Jackson Laboratories (Bar Harbor, USA) and Harlan UK (Bicester, UK), respectively. All mice were housed in individually ventilated cages in a specific pathogen-free environment with a 12 h light-dark cycle and were fed a regular unrestricted diet. The GBF's routine surveillance program screened for selected pathogens. The Ptdsr-deficient progeny. Females were daily monitored for vaginal plugs, and noon of the day of plug detection was defined as E0.5. Embryos at indicated time points were dissected in sterile PBS, washed in ice-cold PBS and transferred to cold fixative. Extra-embryonic membranes were kept and used for genotyping. Ptdsr -/- embryos and their wild-type littermates were used for experiments.Heterozygous male and female mice were intercrossed in order to obtain Ptdsr -/- specimens were used for immunohistochemistry. For detection of apoptotic cells and macrophages, anti-aCasp3  and anti-F4/80  antibodies were used as described by the supplier. Detection was performed using indirect streptavidin with biotinylated secondary antibodies and cobalt-enhanced diaminobenzidine (brown) or fast-red (red) as chromogens. Sections were counterstained with hematoxylin. For whole-mount terminal deoxynucleotidyl transferase-mediated UTP end labeling (TUNEL), limb buds were dissected from E12.5 and E13.5 embryos, fixed in 4% paraformaldehyde and processed for analysis as previously described [Embryos for histology and immunohistochemistry were harvested and fixed in 10% neutral-buffered formalin, dehydrated through a graded series of alcohol, embedded in paraffin, sagittally sectioned at 5 μm intervals, and every fifth section was processed for hematoxylin and eosin (H&E) staining according to standard protocols. Remaining sections of wild-type and escribed .g; 3 min) cells were resuspended in X-Vivo 15 medium supplemented with 50 ng/ml macrophage colony-stimulating factor  and cultured on non-treated tissue-culture dishes at 37°C with 5% CO2. Every second or third day the medium was changed by centrifugation. Following withdrawal of M-CSF on day 6 after excision, adherent cells were cultured for an additional 24-48 h in X-Vivo 15 medium.Fetal livers were excised from embryos at E12.5 and E13.5, respectively, washed in PBS and dissociated enzymatically for 60 min at 37°C. The digestion buffer (150 μl per liver) comprised 0.6 U/ml dispase I (Roche), 0.1% collagenase D (Roche), 10 U DNase (Roche), and 20% FCS in PBS. X-Vivo 15 medium (Cambrex, East Rutherford, USA) was added to the resulting cell suspension, and after centrifugation (200 × 5 cells per well) in X-Vivo 15 medium. For preparation of apoptotic target cells, primary thymocytes were harvested from the thymus of 4- to 8-week-old C57BL/6J mice, stained with TAMRA for 15 min, and apoptosis was induced either by treating cells with 5 μM staurosporine in medium for 4 h at 37°C or by culturing cells in medium overnight. The efficacy of apoptosis induction was compared in thymic target cells and controls by FACS analysis. On average, 60% of the cells of the resulting population were apoptotic, with exposed PS on their surface, and less than 5% of the cells were necrotic, as confirmed by FITC-annexin V and propidium iodide staining. The apoptotic thymocytes obtained were washed with PBS and added to the prepared FLDM cultures (ratio 10:1). Phagocytosis was then allowed to proceed at 37°C and 5% CO2. After the indicated time periods, the uptake of apoptotic cells by FLDMs was stopped by intensive washing of co-cultures with cold PBS to remove unphagocytosed cells. To measure phagocytosis of apoptotic thymocytes, macrophages were further processed for immunofluorescence analysis. Cells were fixed in 4% paraformaldehyde, blocked in 0.5% BSA/PBS and stained with an anti-F4/80 antibody (Serotec) followed by a secondary antibody coupled to Alexa 488 (Molecular Probes Inc., Eugene, USA). Coverslips were mounted on slides and engulfed thymocytes were enumerated by fluorescence microscopy. The percentage of phagocytosis was calculated by counting at least 300 macrophages and determining the number of macrophages that had engulfed apoptotic thymocytes. The phagocytotic index was calculated according to the following formula: phagocytotic index =  ×  × 100. The experiments were performed at least three times, each time in triplicate, and the counting was done by three different investigators.For preparation of monolayer cultures of macrophages, FLDMs were plated on glass coverslips in 24 well plates , apoptotic cells (ratio 1:10) or both for the determination of IL-10, TGF-β1 or TNF-α levels after co-culture for 22 h. For TNF-α quantification at various time points, FLDMs were cultured with a high concentration of LPS (100 ng/ml). Culture supernatants were harvested and TNF-α  and TGF-β1  were measured by ELISA as described by the supplier. IL-10 in culture supernatants was determined by a cytometric bead assay  as indicated in the manual. Data are presented as mean ± SEM from at least three independent experiments, each carried out in triplicate. Analysis of the results used the Wilcoxon-signed rank test; Ptdsr-knockout mice.Additional data file Ptdsr-knockout miceFigure S1 showing the localization of apoptotic cells and macrophages in the subcutis of developing embryos; and Figure S2 showing immunohistochemical staining of the Ptdsr protein in macrophages derived from wild-type and Click here for additional data file


It directly reacts with reactive oxygen species (ROS), functions as a cofactor of antioxidant enzymes, and maintains thiol redox potential in cells. GSH depletion has been implicated in the pathogenesis of neurological diseases, particularly to Parkinson's disease (PD). The purpose of this study was to investigate the change of cellular antioxidant status and basic cell functions in the relatively early stages of GSH depletion.in this study, GSH was depleted by inhibition of glutamylcysteine synthetase using buthionine sulfoximine (BSO) treatment in Ht22, a neuronal cell line derived from mouse hippocampus. Treatment with BSO produced dose-dependent decreases in total GSH level, Fe3+-reducing ability (FRAP assay), Cu2+-reducing ability , and ABTS free radical scavenging ability (ABTS assay) of the cells, but the sensitivity of these indicators to dosage varied considerably. Most of the changes were completed during the first 8 hours of treatment. Cell viability was tested by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromid) assay, and cells at lower density in culture were found to be more sensitive to GSH depletion. The activity of antioxidant enzymes, such as glutathione peroxidase (GPx), glutathione reductase (GR), and copper/zinc superoxide dismutase (Cu/Zn-SOD) were affected by GSH depletion. A cDNA expression array assay of the effects of BSO treatment showed significantly decreased mRNA level for 3 genes, and significantly increased mRNA level for 10 genes, including the antioxidant enzymes Cu/Zn-SOD and thioredoxin peroxidase 2 (TPxII).the study suggests that there are BSO-sensitive and BSO-resistant pools of GSH in Ht22 cells, and that different categories of antioxidant react differently to GSH depletion. Further, the effect of GSH status on cell viability is cell density dependent. Finally, the alterations in expression or activity of several antioxidant enzymes provide insight into the various cellular responses to GSH depletion. Glutathione (GSH, tripeptide γ-L-glutamyl-L-cysteinyl-glycine) is the most abundant thiol antioxidant in mammalian cells. It reacts directly with reactive oxygen species (ROS), or functions as a cofactor of antioxidant enzymes such as the glutathione peroxidases (GPxs). In addition, GSH keeps sulfhydryl groups of cytosolic proteins in reduced form by maintaining thiol redox potential in cells , and regBrain consumes 20% of total oxygen in the body, and thus undergoes high levels of oxidative challenge. Cumulative oxidative damage has been strongly implicated in neurodegeneration and neurological diseases, such as Parkinson's disease (PD), Alzheimer's disease (AD), and amyotrophic lateral sclerosis (ALS). The importance of GSH in particular has been indicated in the case of PD. Among the progressive oxidative stresses that occur in the pathogenesis of PD, an characteristic is the decrease in total GSH concentrations in the substantia nigra in preclinical stages, when other biochemical changes are still undetectable .Investigation of the consequences of intracellular GSH depletion in neuronal cell lines has relied predominantly on one of three methods. These are: (a) treatment with homocysteic acid (HCA) or glutamate to block the uptake of cystine, a substrate for GSH synthesis -7; (b) tIn this study, GSH was depleted by BSO in Ht22, a neuronal cell line derived from mouse hippocampus. GSH level, TAC, antioxidant enzyme activity, cell viability and gene expression were assessed.Before depleting GSH from cells, total GSH levels in Ht22 were monitored from pre-log phase to the end of log phase of cell growth Figures  &2. IntrWhether the decrease of GSH with cell growth is due to limited nutrient supply or to programmed regulation is not known. It has been reported that the GSH content of brain cells depends strongly on the availability of precursors for GSH . It was The dose responses of Ht22 to BSO were analyzed by varying BSO concentration from 0.03 to 10 mM in a 15-hr treatment, and the changes of intracellular GSH level and TAC were measured ; only about 5% of the total glutathione was found in the oxidized form (GSSG). After 15 hrs BSO treatment, malondialdehyde (MDA) assay showed the increases of Abs at 586 nm caused by MDA formation were: control = 0.011 ± 0.006; 1 mM BSO = 0.012 ± 0.006; 3 mM BSO = 0.009 ± 0.004; and 10 mM BSO = 0.011 ± 0.004 (average ± SD, 3–5 independent experiments), no increase in lipid peroxidation after the treatments was observed, indicating GSH depletion at these levels was not yet destructive to the cells.Three methods were employed in this study to investigate antioxidant status following BSO treatment of Ht22 cells. The Cu2+-reducing ability assay  and Fe3+-reducing ability assay (FRAP) both measure the activity of metal ion-reducing antioxidants, but the FRAP assay is characterized by its low pH (3.7), thus excluding the antioxidant function of thiols. Figure In contrast to FRAP and AOP, the ABTS free radical scavenging ability (ABTS assay) of the cells maintained at about 80% of the control level at all BSO concentrations. Cao et al.  previousEffects of GSH depletion on cell viability were assayed using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromid)-based cell viability assay, an indicator of mitochondrial activity  for untreated cells, and 99.6% (N = 5), 99.1% (N = 5), and 98.1% (N = 12), after 15 hrs treatment with 1, 5, and 10 mM BSO, respectively. The oxidative stress produced in these treatments is not sufficient to be lethal to the cells, in accordance with the MDA assay, which shows no increase in lipid peroxidation.Figure The responses of Ht22 cells to 0.1 mM BSO treatment were tested at 4, 8, 12 and 15 hrs, and results are expressed as percentage of 0 hr Figure . For totSeyfried et al.  found thGlutathione peroxidases (GPxs) are selenium-containing antioxidant enzymes that reduce hydrogen peroxide to water, or lipid peroxides to ethanols, with GSH as reducing cofactor. Five isoforms of the GPx family are commonly known. In this study, the major activity detected by the assay is cellular GPx (GPx1). Figure Glutathione reductase (GR) reduces GSSG to GSH using NADPH as cofactor. When Ht22 cells were subjected to 1, 3 and 10 mM BSO treatment for 15 hrs, the GR activity in the cells dropped to 97%, 95% and 94% of control, respectively Figure . At 10 mSuperoxide dismutase (SOD) converts superoxide anion to hydrogen peroxide, which can be further detoxified by GPx1 or catalase. Two isoforms of SOD are found in mammalian cells, known as Cu/Zn-SOD and Mn-SOD, and localized in cytosol and mitochondria, respectively. Incubating Ht22 cells with 1, 3, and 10 mM BSO for 15 hrs increased the Cu/Zn-SOD activity to 104%, 112%, and 152% P= 0.0619) of the control level  to monitor the changes in gene expression after 15 hrs treatment with 10 mM BSO. Out of a total of 465 genes in the array, mRNA level of 3 genes were significantly decreased, and 10 significantly increased  in 48-well cell culture plates (Costar) 12 hrs before BSO treatment. For other assays, the cells were allowed to reach 80–90% of optical confluency (about 2e5 cells per square cm) in 100 mm cell culture dishes (Costar) before the BSO treatment.Ht22 cells were fed with Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) supplement, and cultured at 50–55% relative humidity (RH), in 5% COIn this study, three methods were employed for total antioxidant capacity assay. The AOP assay  tests the ability of samples to reduce Cu2+ to Cu+ at physiological pH, (assayed according to manufacturer's instructions with minor adjustments). The FRAP assay  tests thA common method of testing cell viability is trypan blue staining. As a vital dye, trypan blue enters dead cells, distinguishing them from live ones. In this study, 0.4% (w/v) trypan blue-PBS solution was mixed with properly diluted cells at 5:1 ratio, and cell numbers were counted using a hemocytometer. Cell viability can also be reflected by dehydrogenase activity, which indicates the activity of mitochondria (Cell growth determination kit/MTT based, Sigma). Dehydrogenase converts MTT into purple MTT formazan, causing a colorimetric change that can be monitored photometrically. The bioreduction activity of cells was monitored by an in vitro toxicology assay kit (Sigma), based on a blue to red color change when the oxidoreduction dye, resazurin, is reduced by the bioreduction activity of the cells. Both MTT and Resazurin assays were carried out following the kit instructions.GPx activity assay was based on the classical principle  with optAtlas rat toxicology array II was purchased from BD, and the assay was carried out following the instruction manual.F-test in SAS procedure "Proc GLM" was used for statistical work.JC designed experiments, treated and harvested samples, optimized and participated in the antioxidant assays, participated in the toxicology cDNA array assay. ASH participated in the toxicology cDNA array assay. AY participated in harvesting samples, and the antioxidant assays. MJB contributed to conception and design, critical revision and final approval of the article.


The objective of this study was to test the primary hypothesis that parent proxy-report of pediatric health-related quality of life (HRQL) would prospectively predict pediatric healthcare costs over a two-year period. The exploratory hypothesis tested anticipated that a relatively small group of children would account for a disproportionately large percent of healthcare costs.317 children (157 girls) ages 2 to 18 years, members of a managed care health plan with prospective payment participated in a two-year prospective longitudinal study. At Time 1, parents reported child HRQL using the Pediatric Quality of Life Inventory™ (PedsQL™ 4.0) Generic Core Scales, and chronic health condition status. Costs, based on health plan utilization claims and encounters, were derived for 6, 12, and 24 months.In multiple linear regression equations, Time 1 parent proxy-reported HRQL prospectively accounted for significant variance in healthcare costs at 6, 12, and 24 months. Adjusted regression models that included both HRQL scores and chronic health condition status accounted for 10.1%, 14.4%, and 21.2% of the variance in healthcare costs at 6, 12, and 24 months. Parent proxy-reported HRQL and chronic health condition status together defined a 'high risk' group, constituting 8.7% of the sample and accounting for 37.4%, 59.2%, and 62% of healthcare costs at 6, 12, and 24 months. The high risk group's per member per month healthcare costs were, on average, 12 times that of other enrollees' at 24 months.While these findings should be further tested in a larger sample, our data suggest that parent proxy-reported HRQL can be used to prospectively predict healthcare costs. When combined with chronic health condition status, parent proxy-reported HRQL can identify an at risk group of children as candidates for proactive care coordination. Predicting healthcare costs for pediatric populations has been challenging . AlthougResearchers working with adult populations have linked health status with several important outcomes. In general populations, self-reported health status has been shown to be a predictor of future health services charges , the usethe most important outcome in child health services research [Increasingly, health-related quality of life (HRQL) has become recognized as an important health outcome, some contend research -18. Reseresearch -27. HRQLresearch , and to research ,29, to cresearch , to severesearch , and to research  and barrresearch . Measuriresearch , in deteresearch , and, atresearch . While sresearch ,36.Consequently, the objective of this study was to test the primary hypothesis that parent proxy-report of pediatric HRQL would prospectively predict pediatric healthcare costs over a two-year period. The exploratory hypothesis tested anticipated that a relatively small group of children would account for a disproportionately large percent of healthcare costs.The study took place in San Diego, California between January 1998 and December 2000. We recruited members of a 50,000-member federally supported (Medicaid) managed care health plan. Additional inclusion criteria were that children be between 2 and 18 years of age and that the parent be able to speak either English or Spanish. We exclude children under 2 years of age because the PedsQL™ does not assess parent proxy-report HRQOL below age 2 years. In order to maximize the heterogeneity of the sample, subjects were recruited from three types of healthcare settings: children presenting at pediatricians' offices for scheduled well-child checks (n = 18, 5.7%), children at one of two hospital specialty clinics – orthopedics (n = 6, 1.9%) and cardiology (n = 7, 2.1%) – or children who had been seen at the hospital or its outpatient clinics at least three months previously (n = 286, 90.3%). The data reported here were collected as part of the initial field test to assess the reliability and validity of the PedsQL™ 4.0 Generic Core Scales [The PedsQL™ 4.0 Generic Core Scales  were desHigher PedsQL™ 4.0 scores indicate better HRQL. To create Scale Scores, the mean is computed as the sum of the items divided by the number of items answered (this accounts for missing data). If more than 50% of the items in the scale are missing, the Scale Score is not computed. Imputing the mean of the completed items in a scale when 50% or more are completed is generally the most unbiased and precise method . BecauseParents were asked to report on the presence of a chronic health condition for their child. They read the following statement: "A chronic health condition is: (1) a physical or mental health condition (2) that has lasted or is expected to last at least 6 months and (3) interferes with your child's activities." They then responded with yes or no to the question "In the past 6 months, has your child had a chronic health condition?" If yes, the parents were asked to identify the name of the chronic health condition. Parents who answered yes or who gave the name of a chronic health condition were coded as having a child with a chronic health condition. This method has been used in previous work ,31, and Healthcare costs were calculated as the dollar amount paid by the health plan per patient. We first determined patients' eligibility from the health plan's eligibility data files for three consecutive cumulative periods: 0–6 months, 0–12 months, and 0–24 months after the date they completed the PedsQL™ 4.0. A pediatric patient was considered eligible for health plan benefits for those periods if they were eligible for at least 5 months out of the 6-month period. We then electronically captured healthcare costs  for each pediatric patient for those periods in which they were eligible. We did this by matching each eligible pediatric patient with the health plan's existing databse of claims and encounter data. These data include the dollar amount spent by the health plan. Healthcare costs included hospital and emergency room costs, professional fees, durable medical equipment, home health, specialty clinic, and primary care costs. We did not have access to pharmacy or mental health costs.In California, the site of the study, treatment for 22 specific diagnoses is "carved out," or paid through a separate program  regardless of a child's health plan membership. Thus, for health plans in California, treatment of CCS-covered diagnoses might not be measured in calculating utilization. However, because California's carve out may differ from other states' methods of financing treatment for these diagnoses, and to more completely describe healthcare costs, we included the costs for procedures covered by CCS in our healthcare costs calculations. To derive these costs, we linked the procedure codes on the health plan's CCS referral with the federally supported health plan's fee schedule. These data thus represent the dollar amount the health plan would have spent had the services not been carved out.A convenience sample – subects were recruited nonsystematically when research assistants were available – was recruited at pediatrician offices and specialty clinics. These pediatric patients were identified through examination of the clinic appointment schedules. At these sites, parents of children identified as possible study participants were informed of the study by one of the research assistants after checking in for their appointment, but before being seen by their healthcare provider. Written informed consent included permission for the researchers to examine the medical record to assess utilization. After written informed consent was obtained, the parent completed the proxy-report version of the PedsQL™ 4.0. The research assistant was available at all times to answer any questions.A random sample was recruited from children and adolescents ages 2–18 years who had been seen as inpatients or outpatients at Children's Hospital and Health Center between April 1 and June 30, 1998, and who were members of the health plan. This sample excluded children with a discharge status of expired, children whose payer was from the victim/witness fund, and children whose parents had requested their phone number and address to be kept private. Research assistants called parents of children on this list and obtained verbal informed consent. The research assistant verbally administered the PedsQL™ 4.0 to parents. This research protocol was approved by the institutional review board at Children's Hospital and Health Center, San Diego (#98-020).R2, a measure of the percent of variance in the dependent variable accounted for by the predictor variables while adjusting for the complexity of the model, and the standardized regression coefficient, or beta, for each predictor.We pooled the data from the two samples. Previous reasearch on the PedsQL™ has documented the lack of mode of administration effects ,20. In oPedsQL™ 4.0 scores were skewed toward the high end of the scale and were transformed by taking the square root of the reverse of the score (sqrt(100-score)) in order to create a more normal distribution. The distribution of cost data was skewed to the lower end, with many children having little cost and a relatively smaller number of children having high costs. These data were normalized by taking the log of the costs.In order to explore whether HRQL and chronic health condition status together would define a relatively small subset of enrollees who accounted for a disproportionately large percent of healthcare costs, we divided the sample into quintiles based on the PedsQL™ 4.0 Physical Functioning Scale score and into two groups based on chronic health condition status. Those children who fell in the lowest PedsQL™ 4.0 quintile and who reported the presence of a chronic health condition were assigned to the high-risk group. We describe the percent of costs, per member costs, and per member per month costs per child accounted for by this high risk group.Data was collected from the parents of 317 children (157 girls, 160 boys) ages 2 to 18 years. The average age of the children was 8.3 years (SD = 4.14) with a range of 2.03 to 17.13 years. The sample was heterogeneous with respect to race/ethnicity, with 76 (25.4%) White non-Hispanic, 155 (51.8%) Hispanic, 39 (13.0%) Black non-Hispanic, 6 (2.0%) Asian/Pacific Islander, 3 (1.0%) American Indian or Alaskan Native, 20 (6.7%) Other, and 19 (6.0%) missing. With respect to mother's education, 36.4% had less than a high school education, 46.6% had a high school diploma or some college, and 7.0% were college graduates or beyond (18.8% missing). The measures were administered in two languages – English (n = 233, 73.6%) and Spanish (n = 84, 26.4%). The sample represented both chronically ill (n = 102, 32.1%) and healthy children (n = 215, 67.9%), based on parent report of the presence of a chronic health condition. Table There were no differences found in PedsQL™ scores between the group sampled at well-child checks or specialty clinics and that sampled by phone.All 317 children were enrolled in the health plan after 6 months, with 314 (99.0%) enrolled after 12 months, and 244 (76.9%) after 24 months. There were no differences between those enrolled versus not enrolled at 24 months in percent with a chronic health condition, race/ethnicity, mother's education, or PedsQL™ scores. The cost per member per month (pmpm) for this sample, which represents the total cost divided by the number of members divided by the number of months enrolled, was $149 at 6 months, $137 at 12 months, and $115 at 24 months.The sample included 4,954 claims  over the 24 months. The largest category of visits was for upper respiratory infections (URIs) and related infections (10.96%). Asthma, other infections, otitis media, and pain each account for 5 to 6% of visits, with acute orthopedic conditions accounting for 2.6% of visits. These most common diagnoses account for more than a third (38.7%) of the visits, the rest is comprised of a large number of relatively low-frequency diagnoses. This distribution of diagnoses is similar to the epidemiology of childhood illness, in that much of pediatric morbidity is accounted for by a large number of relatively low frequency diagnoses ,40.Table Table We used the two variables accounting for most of the variance in the regression analysis – the PedsQL™ 4.0 Physical Functioning scores and chronic health condition status – to describe the percentage of costs accounted for by different groups of children. In order to create a single denominator for the percentages, we used the 241 children continuously enrolled in the health plan with complete data for this set of analyses. To create quintiles, we determined the values that divided the sample into five equal-sized groups based on PedsQL™ 4.0 Physical Functioning Scale scores. Enrollees with a score of less than 75 on the PedsQL™ 4.0's 0–100 scale were in the first quintile . The second quintile  was bounded by the scores 75.0 to 90.624, the third quintile  by the scores 90.625 to 96.874, the fourth quintile by the scores 96.875 to 100 , and the fifth quintile consisted of enrollees scoring 100 . Because the distribution of these PedsQL™ 4.0 scores was skewed, we combined the fourth and fifth quintiles .Table Table This study tested the primary hypothesis that HRQL could prospectively predict healthcare cost in pediatric patients in a managed care environment. We measured age, chronic health condition status, and PedsQL™ 4.0 scores at Time 1, and prospectively measured utilization, via costs based on claims and encounter data, for three cumulative periods. These data demonstrate that parent-reported HRQL, as measured by the PedsQL™ 4.0, and chronic health condition status each accounted for significant variance in healthcare costs over 6, 12, and 24 months. The data further show how these two predictor variables, chronic health condition status, and PedsQL™ 4.0 Physical Functioning scores, define a relatively small group of enrollees that accounted for a large percentage of total healthcare costs.This high risk group displays disproportionately high costs as early as 6 months, and their pmpm costs peak at one year. This suggests the importance of managing high risk enrollees as soon as they are identified, perhaps as early as their initial enrollment. It also implies the potential for significant return on investment for better case management, even in the first six months of enrollment. The high risk group's costs remain disproportionately high throughout the 24 months of the study. This fact suggests that the method used here for identifying the high risk group succeeded in identifying children with high ongoing care needs and costs, as opposed to children with one-time health care needs. An anomalous finding was that children in the third quintile on PedsQL™ scores who had chronic health conditions were, for an unexplained reason, much less costly than their peers.It is worth comparing the mean PedsQL™ 4.0 scores for the high risk group to other published data. The high risk group had scores of 44.5 for the Physical Functioning Scale, 70.7 for the Psychosocial Summary Scale, and 61 for the Total Scale. This is placed in clinical perspective by other data showing that scores for children with cancer, in active treatment, are 65, 68, and 67 for the Physical, Psychosocial and Total scales, respectively. .A hypothetical example is presented to illustrate the potential impact of these findings. In a typical health plan, the rate of chronic health conditions will most likely be between 5%  and 18% Taken together, these findings represent an alternative method toward the prospective prediction of healthcare costs in pediatric federally supported managed care populations. While a percentage of these identified costs are inevitable, due to the costs of appropriate care for these chronically ill, poorly functioning children, the possibility exist that a proportion of these healthcare costs are avoidable. Evidence-based disease management has been shown to reduce healthcare costs and increase HRQL in certain chronic conditions such as asthma ,44. By iCertain limitations exist in this study. The first has to do with data not accounted for in this study. We did not have access to pharmacy and mental health costs, nor did we have access to out-of-plan expenditures. For mental health costs, however, recent data has shown that children referred for psychiatric services demonstrate child self-report and parent proxy-report PedsQL™ 4.0 Total Scale Scores comparable to children with chronic physical health conditions . Those dThe second limitation has to do with sampling issues and with generalizing these data beyond this study. Our data was not collected at enrollment, nor did we sample from the entire pool of enrollees. Further research is necessary to determine whether these findings hold true for children assessed at health plan enrollment, and to determine the extent to which the results may be influenced by the convenience sample used here. The sample was too small to use cross-validation techniques. Prediction models tend to overfit the development sample, and the predictive validity of these variables should be tested in other, larger samples. Although generalization of these findings to broader populations should be made with caution, the sample here is very diverse with respect to race/ethnicity, and thus likely to be similar to other federally support health plans. We also combined data from two different samples – specialty clinic patients, and health plan members who had been seen in the hospital or outpatient clinics at least three months after the clinical encounter. These two groups could have had unmeasured systematic differences, which could have biased the results.The third has to do with using a survey to gather these data. The costs of fielding a survey can be quite high, and, if tied to payment, survey responses are subject to "gaming". However, we submit that the potential gains from optimal management of an enrolled population will almost certainly be greater than the costs of survey administration. Moreover, while gaming might occur if health plans were to be compensated based on the HRQL of their enrolled population, the methods used here are suggested as strategies for clinical management, not for rate setting, thus reducing the incentives for gaming. We did not track refusal rates and so do not know what percent of potential participants consented to be in the study. Finally, using a survey means that parents reported on their children's chronic health condition information. Objective measures of chronic health condition would strengthen the validation process. However, in previous PedsQL™ 4.0 clinical research in pediatric patients with cancer, cardiac and rheumatic chronic health conditions, objective medical diagnosis of these chronic diseases demonstrated similar differences between healthy children and children with chronic health conditions as shown in the present findings -30.Further research is necessary. First, a much larger, and randomly selected sample is necessary to confirm these results. Second, split-half validation should be performed so that the coefficients from one group are used to predict the costs in a different group. This could be done with split halves of one large group, or with two similar groups enrolled at different points in time. Given that our regression equation explains 21% of costs, further studies could be done to determine whether other variables might account for additional variance in costs. Further studies could also allow comparison and validation with the results here.This is the first study we are aware of to use parent reports of pediatric HRQL and chronic health condition status to prospectively predict healthcare costs in a pediatric sample. These data have implications for healthcare decision makers such as pediatricians, health plan administrators, and policymakers. In a prospective payment system, providers are incentivized to actively manage high-risk patients and to provide care at the appropriate level. The idea behind such a system is that prevention and appropriate care accrues benefits to patients in the form of better health and to providers in the form of lower costs. If, as these data suggest, parent reports of HRQL can be used to predict healthcare costs, one could identify at-risk children proactively and intervene to avoid both illness and costs. In this way, these data can serve simultaneously to improve the health of children and the system that serves them.HRQL Health-related Quality of LifePedsQL™ 4.0 Pediatric Quality of Life Inventory™, Version 4.0SD Standard DeviationCCS California Children's ServicesURI Upper respiratory infectionPMPM per member per monthMS, PSK, and JWV conceived of the research. MS and JWV supervised the data collection. MS and DS performed the data analysis. MS drafted the manuscript. JWV, PSK, and DS provided substantive input into the manuscript. All authors read and approved the final manuscript.