Melanoma antigens in pediatric medulloblastoma contribute to tumor heterogeneity and species-specificity of group 3 tumors.

Background: Medulloblastoma (MB) is the most malignant childhood brain cancer. Group 3 MB subtype accounts for about 25% of MB diagnoses and is associated with the most unfavorable outcomes. Herein, we report that more than half of group 3 MB tumors express melanoma antigens (MAGEs), which are potential prognostic and therapeutic markers. MAGEs are tumor antigens, expressed in several types of adult cancers and associated with poorer prognosis and therapy resistance; however, their expression in pediatric cancers is mostly unknown. The aim of this study was to determine whether MAGEs are activated in pediatric MB. Methods: To determine MAGE frequency in pediatric MB, we obtained formalin-fixed paraffin-embedded tissue (FFPE) samples of 34 patients, collected between 2008 - 2015, from the Children's Medical Center Dallas pathology archives and applied our validated reverse transcription quantitative PCR (RT-qPCR) assay to measure the relative expression of 23 MAGE cancer-testis antigen genes. To validate our data, we analyzed several published datasets from pediatric MB patients and patient-derived orthotopic xenografts, totaling 860 patients. We then examined how MAGE expression affects the growth and oncogenic potential of medulloblastoma cells by CRISPR-Cas9- and siRNA-mediated gene depletion. Results: Our RT-qPCR analysis suggested that MAGEs were expressed in group 3/4 medulloblastoma. Further mining of bulk and single-cell RNA-sequencing datasets confirmed that 50-75% of group 3 tumors activate a subset of MAGE genes. Depletion of MAGEAs, B2, and Cs alter MB cell survival, viability, and clonogenic growth due to decreased proliferation and increased apoptosis. Conclusions: These results indicate that targeting MAGEs in medulloblastoma may be a potential therapeutic option for group 3 medulloblastomas. Key Points: Several Type I MAGE CTAs are expressed in >60% of group 3 MBs. Type I MAGEs affect MB cell proliferation and apoptosis. MAGEs are potential biomarkers and therapeutic targets for group 3 MBs. Importance of the Study: This study is the first comprehensive analysis of all Type I MAGE CTAs ( MAGEA , -B , and -C subfamily members) in pediatric MBs. Our results show that more than 60% of group 3 MBs express MAGE genes, which are required for the viability and growth of cells in which they are expressed. Collectively, these data provide novel insights into the antigen landscape of pediatric MBs. The activation of MAGE genes in group 3 MBs presents potential stratifying and therapeutic options.

Synaptic plasticity in human thalamocortical assembloids.

Synaptic plasticities, such as long-term potentiation (LTP) and depression (LTD), tune synaptic efficacy and are essential for learning and memory. Current studies of synaptic plasticity in humans are limited by a lack of adequate human models. Here, we modeled the thalamocortical system by fusing human induced pluripotent stem cell-derived thalamic and cortical organoids. Single-nucleus RNA-sequencing revealed that most cells in mature thalamic organoids were glutamatergic neurons. When fused to form thalamocortical assembloids, thalamic and cortical organoids formed reciprocal long-range axonal projections and reciprocal synapses detectable by light and electron microscopy, respectively. Using whole-cell patch-clamp electrophysiology and two-photon imaging, we characterized glutamatergic synaptic transmission. Thalamocortical and corticothalamic synapses displayed short-term plasticity analogous to that in animal models. LTP and LTD were reliably induced at both synapses; however, their mechanisms differed from those previously described in rodents. Thus, thalamocortical assembloids provide a model system for exploring synaptic plasticity in human circuits.

Chemical and Genetic Modulation of Complex I of the Electron Transport Chain Enhances the Biotherapeutic Protein Production Capacity of CHO Cells.

Chinese hamster ovary (CHO) cells are the cell line of choice for producing recombinant therapeutic proteins. Despite improvements in production processes, reducing manufacturing costs remains a key driver in the search for more productive clones. To identify media additives capable of increasing protein production, CHOZN® GS-/- cell lines were screened with 1280 small molecules, and two were identified, forskolin and BrdU, which increased productivity by ≥40%. While it is possible to incorporate these small molecules into a commercial-scale process, doing so may not be financially feasible or could raise regulatory concerns related to the purity of the final drug substance. To circumvent these issues, RNA-Seq was performed to identify transcripts which were up- or downregulated upon BrdU treatment. Subsequent Reactome pathway analysis identified the electron transport chain as an affected pathway. CRISPR/Cas9 was utilized to create missense mutations in two independent components of the electron transport chain and the resultant clones partially recapitulated the phenotypes observed upon BrdU treatment, including the productivity of recombinant therapeutic proteins. Together, this work suggests that BrdU can enhance the productivity of CHO cells by modulating cellular energetics and provides a blueprint for translating data from small molecule chemical screens into genetic engineering targets to improve the performance of CHO cells. This could ultimately lead to more productive host cell lines and a more cost-effective method of supplying medication to patients.

Exploration of overlap volumes for radiotherapy plan evaluation with the aim of healthy tissue sparing.

PURPOSE: Development of a novel interactive visualization approach for the exploration of radiotherapy treatment plans with a focus on overlap volumes with the aim of healthy tissue sparing. METHODS: We propose a visualization approach to include overlap volumes in the radiotherapy treatment plan evaluation process. Quantitative properties can be interactively explored to identify critical regions and used to steer the visualization for a detailed inspection of candidates. We evaluated our approach with a user study covering the individual visualizations and their interactions regarding helpfulness, comprehensibility, intuitiveness, decision-making and speed. RESULTS: A user study with three domain experts was conducted using our software and evaluating five data sets each representing a different type of cancer and location by performing a set of tasks and filling out a questionnaire. The results show that the visualizations and interactions help to identify and evaluate overlap volumes according to their physical and dose properties. Furthermore, the task of finding dose hot spots can also benefit from our approach. CONCLUSIONS: The results indicate the potential to enhance the current treatment plan evaluation process in terms of healthy tissue sparing.

Analytical performance of the FDA-cleared Parsortix® PC1 system.

Introduction: The Parsortix® PC1 system, Food and Drug Administration (FDA) cleared for use in metastatic breast cancer (MBC) patients, is an epitope-independent microfluidic device for the capture and harvest of circulating tumor cells from whole blood based on cell size and deformability. This report details the analytical characterization of linearity, detection limit, precision, and reproducibility for this device. Methods: System performance was determined using K2-EDTA blood samples collected from self-declared healthy female volunteers (HVs) and MBC patients spiked with prelabeled cultured breast cancer cell lines (SKBR3, MCF7, or Hs578T). Samples were processed on Parsortix® PC1 systems and captured cells were harvested and enumerated. Results: The system captured and harvested live SKBR3, MCF7, and Hs578T cells and fixed SKBR3 cells linearly between 2 and ~100 cells, with average harvest rates of 69%, 73%, 79%, and 90%, respectively. To harvest ≥1 cell ≥95% of the time, the system required 3, 5 or 4 live SKBR3, MCF7 or Hs578T cells, respectively. Average harvest rates from precision studies using 5, 10, and ~50 live cells spiked into blood for each cell line ranged from 63.5% to 76.2%, with repeatability and reproducibility percent coefficient of variation (%CV) estimates ranging from 12.3% to 32.4% and 13.3% to 34.1%, respectively. Average harvest rates using ~20 fixed SKBR3 cells spiked into HV and MBC patient blood samples were 75.0% ± 16.1% (%CV = 22.3%) and 68.4% ± 14.3% (%CV = 21.1%), respectively. Conclusions: These evaluations demonstrate the Parsortix® PC1 system linearly and reproducibly harvests tumor cells from blood over a range of 1 to ~100 cells.

Spatial mapping of cellular senescence: emerging challenges and opportunities.

Cellular senescence is a well-established driver of aging and age-related diseases. There are many challenges to mapping senescent cells in tissues such as the absence of specific markers and their relatively low abundance and vast heterogeneity. Single-cell technologies have allowed unprecedented characterization of senescence; however, many methodologies fail to provide spatial insights. The spatial component is essential, as senescent cells communicate with neighboring cells, impacting their function and the composition of extracellular space. The Cellular Senescence Network (SenNet), a National Institutes of Health (NIH) Common Fund initiative, aims to map senescent cells across the lifespan of humans and mice. Here, we provide a comprehensive review of the existing and emerging methodologies for spatial imaging and their application toward mapping senescent cells. Moreover, we discuss the limitations and challenges inherent to each technology. We argue that the development of spatially resolved methods is essential toward the goal of attaining an atlas of senescent cells.

Community-Engaged Assessment of Soil Lead Contamination in Atlanta Urban Growing Spaces.

Urban agriculture is emerging as a method to improve food security and public health in cities across the United States. However, an increased risk of exposure to heavy metals and metalloids (HMM) exists through interaction with contaminated soil. Community-engaged research (CEnR) is one method that can promote the inclusion of all partners when studying exposures such as HMM in soil. Researchers and community gardeners co-designed this study to measure the concentrations of lead (Pb), using X-Ray Fluorescence (XRF) verified with Inductively Coupled Plasma-Mass Spectrometry (ICP-MS) in soils from 19 urban agricultural and residential sites in the Westside of Atlanta and three rural sites in Georgia. Seventeen other HMM were measured but not included in this study, because they did not pose risks to the community comparable to elevated Pb levels. Pb concentrations were compared to the Environmental Protection Agency (EPA)'s regional screening levels (RSLs) for residential soil and the University of Georgia (UGA) extension service's low-risk levels (LRLs) for agriculture. Soils from the majority of sites had levels below EPA RSLs for Pb, yet above the UGA LRL. However, soil Pb concentrations were three times higher than the EPA RSL on some sites that contained metal refining waste or slag. Our findings led to direct action by local and federal government agencies to initiate the cleanup of slag residue. Studies involving exposures to communities should engage those affected throughout the process for maximum impact.

NSD1 mediates antagonism between SWI/SNF and polycomb complexes and is required for transcriptional activation upon EZH2 inhibition.

Disruption of antagonism between SWI/SNF chromatin remodelers and polycomb repressor complexes drives the formation of numerous cancer types. Recently, an inhibitor of the polycomb protein EZH2 was approved for the treatment of a sarcoma mutant in the SWI/SNF subunit SMARCB1, but resistance occurs. Here, we performed CRISPR screens in SMARCB1-mutant rhabdoid tumor cells to identify genetic contributors to SWI/SNF-polycomb antagonism and potential resistance mechanisms. We found that loss of the H3K36 methyltransferase NSD1 caused resistance to EZH2 inhibition. We show that NSD1 antagonizes polycomb via cooperation with SWI/SNF and identify co-occurrence of NSD1 inactivation in SWI/SNF-defective cancers, indicating in vivo relevance. We demonstrate that H3K36me2 itself has an essential role in the activation of polycomb target genes as inhibition of the H3K36me2 demethylase KDM2A restores the efficacy of EZH2 inhibition in SWI/SNF-deficient cells lacking NSD1. Together our data expand the mechanistic understanding of SWI/SNF and polycomb interplay and identify NSD1 as the key for coordinating this transcriptional control.

DIAPH1 Variants in Non-East Asian Patients With Sporadic Moyamoya Disease.

Importance: Moyamoya disease (MMD), a progressive vasculopathy leading to narrowing and ultimate occlusion of the intracranial internal carotid arteries, is a cause of childhood stroke. The cause of MMD is poorly understood, but genetic factors play a role. Several familial forms of MMD have been identified, but the cause of most cases remains elusive, especially among non-East Asian individuals. Objective: To assess whether ultrarare de novo and rare, damaging transmitted variants with large effect sizes are associated with MMD risk. Design, Setting, and Participants: A genetic association study was conducted using whole-exome sequencing case-parent MMD trios in a small discovery cohort collected over 3.5 years (2016-2019); data were analyzed in 2020. Medical records from US hospitals spanning a range of 1 month to 1.5 years were reviewed for phenotyping. Exomes from a larger validation cohort were analyzed to identify additional rare, large-effect variants in the top candidate gene. Participants included patients with MMD and, when available, their parents. All participants who met criteria and were presented with the option to join the study agreed to do so; none were excluded. Twenty-four probands (22 trios and 2 singletons) composed the discovery cohort, and 84 probands (29 trios and 55 singletons) composed the validation cohort. Main Outcomes and Measures: Gene variants were identified and filtered using stringent criteria. Enrichment and case-control tests assessed gene-level variant burden. In silico modeling estimated the probability of variant association with protein structure. Integrative genomics assessed expression patterns of MMD risk genes derived from single-cell RNA sequencing data of human and mouse brain tissue. Results: Of the 24 patients in the discovery cohort, 14 (58.3%) were men and 18 (75.0%) were of European ancestry. Three of 24 discovery cohort probands contained 2 do novo (1-tailed Poisson P = 1.1 × 10-6) and 1 rare, transmitted damaging variant (12.5% of cases) in DIAPH1 (mammalian diaphanous-1), a key regulator of actin remodeling in vascular cells and platelets. Four additional ultrarare damaging heterozygous DIAPH1 variants (3 unphased) were identified in 3 other patients in an 84-proband validation cohort (73.8% female, 77.4% European). All 6 patients were non-East Asian. Compound heterozygous variants were identified in ena/vasodilator-stimulated phosphoproteinlike protein EVL, a mammalian diaphanous-1 interactor that regulates actin polymerization. DIAPH1 and EVL mutant probands had severe, bilateral MMD associated with transfusion-dependent thrombocytopenia. DIAPH1 and other MMD risk genes are enriched in mural cells of midgestational human brain. The DIAPH1 coexpression network converges in vascular cell actin cytoskeleton regulatory pathways. Conclusions and Relevance: These findings provide the largest collection to date of non-East Asian individuals with sporadic MMD harboring pathogenic variants in the same gene. The results suggest that DIAPH1 is a novel MMD risk gene and impaired vascular cell actin remodeling in MMD pathogenesis, with diagnostic and therapeutic ramifications.

Analysis workflow to assess de novo genetic variants from human whole-exome sequencing.

Here, we present a protocol to analyze de novo genetic variants derived from the whole-exome sequencing (WES) of proband-parent trios. We provide stepwise instructions for using existing pipelines to call de novo mutations (DNMs) and determine whether the observed number of such mutations is enriched relative to the expected number. This protocol may be extended to any human disease trio-based cohort. Cohort size is a limiting determinant to the discovery of high-confidence pathogenic DNMs. For complete details on the use and execution of this protocol, please refer to Dong et al. (2020).

In Vivo CRISPR/Cas9-Based Targeted Disruption and Knockin of a Long Noncoding RNA.

The CRISPR/Cas9 system has been widely used as an efficient genome-editing tool for studying physiological functions of long noncoding RNAs (lncRNAs). In this chapter, we describe the experimental procedures for using the CRISPR/Cas9 system to genetically modify a long noncoding RNA in vivo through the targeted disruption and knockin approaches.

Ablating Tau Reduces Hyperexcitability and Moderates Electroencephalographic Slowing in Transgenic Mice Expressing A53T Human α-Synuclein.

Abnormal intraneuronal accumulation of the presynaptic protein α-synuclein (α-syn) is implicated in the etiology of dementia with Lewy bodies (DLB) and Parkinson's disease with dementia (PDD). Recent work revealed that mice expressing human α-syn with the alanine-53-threonine (A53T) mutation have a similar phenotype to the human condition, exhibiting long-term potentiation deficits, learning and memory deficits, and inhibitory hippocampal remodeling, all of which were reversed by genetic ablation of microtubule-associated protein tau. Significantly, memory deficits were associated with histological signs of network hyperactivity/seizures. Electrophysiological abnormalities are often seen in parkinsonian dementias. Baseline electroencephalogram (EEG) slowing is used as a supportive diagnostic feature in DLB and PDD, and patients with these diseases may exhibit indicators of broad network dysfunction such as sleep dysregulation, myoclonus, and seizures. Given the translational significance, we examined whether human A53T α-syn expressing mice exhibit endogenous-tau-dependent EEG abnormalities, as measured with epidural electrodes over the frontal and parietal cortices. Using template-based waveform sorting, we determined that A53T mice have significantly high numbers of epileptiform events as early as 3-4 months of age and throughout life, and this effect is markedly attenuated in the absence of tau. Epileptic myoclonus occurred in half of A53T mice and was markedly reduced by tau ablation. In spectral analysis, tau ablation partially reduced EEG slowing in 6-7 month transgenic mice. We found abnormal sleeping patterns in transgenic mice that were more pronounced in older groups, but did not find evidence that this was influenced by tau genotype. Together, these data support the notion that tau facilitates A53T α-syn-induced hyperexcitability that both precedes and coincides with associated synaptic, cognitive, and behavioral effects. Tau also contributes to some aspects of EEG slowing in A53T mice. Importantly, our work supports tau-based approaches as an effective early intervention in α-synucleinopathies to treat aberrant network activity.

Atlanta Residents' Knowledge Regarding Heavy Metal Exposures and Remediation in Urban Agriculture.

Urban agriculture and gardening provide many health benefits, but the soil is sometimes at risk of heavy metal and metalloid (HMM) contamination. HMM, such as lead and arsenic, can result in adverse health effects for humans. Gardeners may face exposure to these contaminants because of their regular contact with soil and consumption of produce grown in urban areas. However, there is a lack of research regarding whether differential exposure to HMM may be attributed to differential knowledge of exposure sources. In 2018, industrial slag and hazardous levels of soil contamination were detected in West Atlanta. We conducted community-engaged research through surveys and follow-up interviews to understand awareness of slag, HMM in soil, and potential remediation options. Home gardeners were more likely to recognize HMM health effects and to cite health as a significant benefit of gardening than community gardeners. In terms of knowledge, participants were concerned about the potential health effects of contaminants in soil yet unconcerned with produce in their gardens. Gardeners' knowledge on sources of HMM exposure and methods for remediation were low and varied based on racial group.

Case Report: Early-Onset Behavioral Variant Frontotemporal Dementia in Patient With Retrotransposed Full-Length Transcript of Matrin-3 Variant 5.

Frontotemporal dementia (FTD) rarely occurs in individuals under the age of 30, and genetic causes of early-onset FTD are largely unknown. The current report follows a 27 year-old patient with no significant past medical history presenting with two years of progressive changes in behavior, rushed speech, verbal aggression, and social withdrawal. MRI and FDG-PET imaging of the brain revealed changes maximally in the frontal and temporal lobes, which along with the clinical features, are consistent with behavioral variant FTD. Next generation sequencing of a panel of 28 genes associated with dementia and amyotrophic lateral sclerosis (ALS) initially revealed a duplication of exon 15 in Matrin-3 (MATR3). Whole genome sequencing determined that this genetic anomaly was, in fact, a sequence corresponding with full-length MATR3 variant 5 inserted into chromosome 12, indicating retrotransposition from a messenger RNA intermediate. To our knowledge, this is a novel mutation of MATR3, as the majority of mutations in MATR3 linked to FTD-ALS are point mutations. Genomic DNA analysis revealed that this mutation is also present in one unaffected first-degree relative and one unaffected second-degree relative. This suggests that the mutation is either a disease-causing mutation with incomplete penetrance, which has been observed in heritable FTD, or a benign variant. Retrotransposons are not often implicated in neurodegenerative diseases; thus, it is crucial to clarify the potential role of this MATR3 variant 5 retrotransposition in early-onset FTD.

Genome editing of HBG1 and HBG2 to induce fetal hemoglobin.

Induction of fetal hemoglobin (HbF) via clustered regularly interspaced short palindromic repeats/Cas9-mediated disruption of DNA regulatory elements that repress γ-globin gene (HBG1 and HBG2) expression is a promising therapeutic strategy for sickle cell disease (SCD) and ß-thalassemia, although the optimal technical approaches and limiting toxicities are not yet fully defined. We disrupted an HBG1/HBG2 gene promoter motif that is bound by the transcriptional repressor BCL11A. Electroporation of Cas9 single guide RNA ribonucleoprotein complex into normal and SCD donor CD34+ hematopoietic stem and progenitor cells resulted in high frequencies of on-target mutations and the induction of HbF to potentially therapeutic levels in erythroid progeny generated in vitro and in vivo after transplantation of hematopoietic stem and progenitor cells into nonobese diabetic/severe combined immunodeficiency/Il2rγ-/-/KitW41/W41 immunodeficient mice. On-target editing did not impair CD34+ cell regeneration or differentiation into erythroid, T, B, or myeloid cell lineages at 16 to 17 weeks after xenotransplantation. No off-target mutations were detected by targeted sequencing of candidate sites identified by circularization for in vitro reporting of cleavage effects by sequencing (CIRCLE-seq), an in vitro genome-scale method for detecting Cas9 activity. Engineered Cas9 containing 3 nuclear localization sequences edited human hematopoietic stem and progenitor cells more efficiently and consistently than conventional Cas9 with 2 nuclear localization sequences. Our studies provide novel and essential preclinical evidence supporting the safety, feasibility, and efficacy of a mechanism-based approach to induce HbF for treating hemoglobinopathies.

Screening of plant growth promotion ability among bacteria isolated from field-grown sorghum under different managements in Brazilian drylands.

Sorghum [Sorghum bicolor (L.) Moench] is a multipurpose grass cultivated in drylands due to its adaptation to drought. However the characteristics of sorghum-associated bacteria are not known in the Brazilian drylands. The aim of this study was to isolate and evaluate the plant growth promotion potential bacteria from field-grown sorghum under two irrigation and manure application levels in a Brazilian semi-arid reagion. Sorghum was irrigated with 3 or 1 mm day-1 and fertilized or not with liquid goat manure. Bacteria were obtained from surface-disinfected roots applying two nitrogen-free semi-solid media. The bacteria were evaluated for the presence of nifH gene, 16S rRNA sequences, calcium-phosphate solubilization, production of auxins and siderophores and for sorghum growth promotion. We obtained 20 out of 24 positive bacteria for nifH. The isolates were classified as in six different genera. All isolates produced auxins "in vitro", six bacteria produced siderophores and three Enterobacteriaceae solubilized calcium-phosphate. At least ten bacteria resulted in the increased total N content in the sorghum shoots, comparable to fertilization with 50 mg N plant-1 week-1 and to inoculation with Azospirillum brasilense Ab-V5. Enterobacter sp. ESA 57 was the best sorghum plant-growth promoting bacteria isolated in this study.

The COPII cargo adapter SEC24C is essential for neuronal homeostasis.

SEC24 family members are components of the coat protein complex II (COPII) machinery that interact directly with cargo or with other adapters to ensure proper sorting of secretory cargo into COPII vesicles. SEC24C is 1 of 4 mammalian SEC24 paralogs (SEC24A-D), which segregate into 2 subfamilies on the basis of sequence homology (SEC24A/SEC24B and SEC24C/SEC24D). Here, we demonstrate that postmitotic neurons, unlike professional secretory cells in other tissues, are exquisitely sensitive to loss of SEC24C. Conditional KO of Sec24c in neural progenitors during embryogenesis caused perinatal mortality and microcephaly, with activation of the unfolded protein response and apoptotic cell death of postmitotic neurons in the murine cerebral cortex. The cell-autonomous function of SEC24C in postmitotic neurons was further highlighted by the loss of cell viability caused by disrupting Sec24c expression in forebrain neurons of mice postnatally and in differentiated neurons derived from human induced pluripotent stem cells. The neuronal cell death associated with Sec24c deficiency was rescued in knockin mice expressing Sec24d in place of Sec24c. These data suggest that SEC24C is a major cargo adapter for COPII-dependent transport in postmitotic neurons in developing and adult brains and that its functions overlap at least partially with those of SEC24D in mammals.

A Survey of Validation Strategies for CRISPR-Cas9 Editing.

The T7 endonuclease 1 (T7E1) mismatch detection assay is a widely used method for evaluating the activity of site-specific nucleases, such as the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system. To determine the accuracy and sensitivity of this assay, we compared the editing estimates derived by the T7E1 assay with that of targeted next-generation sequencing (NGS) in pools of edited mammalian cells. Here, we report that estimates of nuclease activity determined by T7E1 most often do not accurately reflect the activity observed in edited cells. Editing efficiencies of CRISPR-Cas9 complexes with similar activity by T7E1 can prove dramatically different by NGS. Additionally, we compared editing efficiencies predicted by the Tracking of Indels by Decomposition (TIDE) assay and the Indel Detection by Amplicon Analysis (IDAA) assay to that observed by targeted NGS for both cellular pools and single-cell derived clones. We show that targeted NGS, TIDE, and IDAA assays predict similar editing efficiencies for pools of cells but that TIDE and IDAA can miscall alleles in edited clones.

Active tracking of rejected dried blood samples in a large program in Nigeria.

AIM: To study the impact of rejection at different levels of health care by retrospectively reviewing records of dried blood spot samples received at the molecular laboratory for human immunodeficiency virus (HIV) early infant diagnosis (EID) between January 2008 and December 2012. METHODS: The specimen rejection rate, reasons for rejection and the impact of rejection at different levels of health care was examined. The extracted data were cleaned and checked for consistency and then de-duplicated using the unique patient and clinic identifiers. The cleaned data were ciphered and exported to SPSS version 19 (SPSS 2010 IBM Corp, New York, United States) for statistical analyses. RESULTS: Sample rejection rate of 2.4% (n = 786/32552) and repeat rate of 8.8% (n = 69/786) were established. The mean age of infants presenting for first HIV molecular test among accepted valid samples was 17.83 wk (95%CI: 17.65-18.01) vs 20.30 wk (95%CI: 16.53-24.06) for repeated samples. HIV infection rate was 9.8% vs 15.9% for accepted and repeated samples. Compared to tertiary healthcare clinics, secondary and primary clinics had two-fold and three-fold higher likelihood of sample rejection, respectively (P < 0.05). We observed a significant increase in sample rejection rate with increasing number of EID clinics (r = 0.893, P = 0.041). The major reasons for rejection were improper sample collection (26.3%), improper labeling (16.4%) and insufficient blood (14.8%). CONCLUSION: Programs should monitor pre-analytical variables and incorporate continuous quality improvement interventions to reduce errors associated with sample rejection and improve patient retention.