Compatibility of Stabilized Whole Blood Products with CD4 Technologies and Their Suitability for Quality Assessment Programs

Background

CD4 T cell enumeration is the most widely used prognostic marker for management of HIV disease. Internal quality control and external quality assessment (EQA) programs are critical to ensure reliability of clinical measurements. The utility of stabilized whole blood products (SWBP) as a test reagent for EQA programs such as Quality Assessment and Standardization for Immunological measures relevant to HIV/AIDS (QASI) program have been demonstrated previously. Since then, several new commercial SWBPs and alternative CD4 enumeration technologies have become available. Seven SWBPs were evaluated on seven different enumeration platforms to determine which product(s) are most suitable for EQA programs that support multiple analytical technologies.

Method

Assessment of SWBPs was based on two criteria: (1) accuracy of CD4 T cell measurements and; (2) stability under sub optimal storage conditions.

Results

Three SWBPs (Multi-Check, StatusFlow and CD4 Count) showed accurate CD4 T-cell absolute count and percentage values across six of the enumeration platforms. All products retain stability up to 18 days at 21–23°C with the exception of Multi-Check-high on FacsCount and Multi-Check-Low and StatusFlow-Low on Pima. One of the products (CD4 Count) retained stability for three days on all platforms tested when stored at 37°C.

Conclusion

This study demonstrated that the characteristics of commercially available SWBPs vary across multiple CD4 platforms. The compatibility of testing panels for EQA programs with multiple analytical platforms needs to be carefully considered, especially in large multiplatform CD4 EQA programs. The selection of a suitable cross-platform SWBP is an increasing challenge as more reagents and platforms are introduced for CD4 T-cell enumeration.     

Comparison of single and dual platform methodologies for the estimation of CD34+ hematopoietic progenitor cells: correlation with colony assay

In this study three assays for the enumeration of CD34+ progenitors were compared: 1) a modified version of the Milan protocol, used in the standard dual-platform format; 2) a dual-platform version of the ISHAGE protocol; 3) the ProCOUNT software version 2.0/ProCOUNT kit. The assays were compared to validate the accuracy of CD34+ cell counts in mobilized peripheral blood (PB), apheresis products (AP), and cord blood (CB). The ProCOUNT protocol uses reference beads for absolute CD34+ cell counting, whereas CD34 counts by other techniques are derived from a separate leukocyte count performed by a hematology analyzer. A good correlation between the ISHAGE and ProCOUNT methods was obtained for estimation of CD34+ counts in PB (n=42 samples analyzed) and AP (n=35)--except for samples having a leukocyte count >25 x 10(9)/L or a CD34 count <0.0025 x 10(9)/L)--while a suboptimal correlation between the methods was observed for CB (n=30). The ProCOUNT system proved to be effective in reducing the variability in CD34+ cell counting and appeared to be useful for intralaboratory methodology standardization. The main disadvantage of the ProCOUNT assay was its inability to calculate CD34 counts in leukopenic samples and in CB samples showing a high erythroblast count. As far as the correlation with hematopoietic colonies is concerned, data collected from apheresis samples showed a good correlation between the three flow cytometry methods and colony-forming unit granulocyte-macrophage (CFU-GM) counts, confirming the value of the flow cytometric test as a real-time, truly predictive test to measure the hematopoietic potential of the graft. In summary, all methods are suitable for enumeration of most PB samples, while the single-platform methodology should be preferred for the analysis of AP and CB. We also found the dual-platform format of the ISHAGE method precise and accurate for the estimation of CD34+ cells from CB samples. Based on these data it can be concluded that the single-platform flow cytometry assay format should be the preferred approach for CD34+ stem cell enumeration in different types of samples.     

Quality control of CD4+ T-lymphocyte enumeration: results from the last 9 years of the United Kingdom National External Quality Assessment Scheme for Immune Monitoring (1993-2001)

The human immunodeficiency virus (HIV) global epidemic has necessitated the routine enumeration of T-lymphocyte subsets, which has created a need for external quality assurance (EQA). The United Kingdom National External Quality Assessment Scheme (UK NEQAS) for Immune Monitoring provides EQA for 296 laboratories in 40 countries. In 1993, UK NEQAS developed and incorporated into its program stabilized whole blood that enables the accurate monitoring of laboratory performance. Overall, the mean interlaboratory coefficient of variation (CV) for percentage CD4(+) T-lymphocyte subset enumeration has fallen from 15% to less than 5%, as a direct result of the increased use of CD45/ side scatter (SSC) gating. Laboratories using alternative gating strategies (i.e., CD45/CD14 or forward scatter [FSC]/SSC) were about 7.4 times more likely to fail an EQA exercise. Furthermore, the adoption of single-platform technology resulted in a reduction of the overall mean interlaboratory CV for absolute CD4(+) T lymphocytes from 56% (prior to the widespread use of single-platform technology) to 9.7%. Individual laboratory deficiencies were also identified using a performance monitoring system and, through re-education by collaboration with the coordinating center, satisfactorily resolved. In conclusion, during the last 9 years, the UK NEQAS for Immune Monitoring program has highlighted the significant technological advances made by laboratories worldwide that undertake lymphocyte subset enumeration.     

Multi-laboratory assay for harmonization of enumeration of viable CD34+ and CD45+ cells in frozen cord blood units

Background aimsIn 2016, specifications for both pre-cryopreserved and post-thawed cord blood were defined in the sixth edition of NetCord Foundation for the Accreditation of Cellular Therapy (FACT) Standards for Cord Blood Banks. However, for several experts, harmonization regarding flow cytometry analysis performed on post-thawed samples is still a concern. A multicenter study led by Héma-Québec aimed to provide scientific data to support the cord blood accreditation bodies such as NetCord FACT in the revision of standards.MethodsTwelve cord blood units were processed for plasma and red cell reduction following standard operating procedures. Cord blood unit aliquots were shipped to eight participating centers under cryogenic conditions for analysis before and after standardization of protocol. Repeatability of stem cell count, measured pre- and post-intervention with the centers, was estimated using multilevel linear regression models with a heterogeneous compound symmetry correlation structure among repeated measures.ResultsExcellent inter-center repeatability was reported by each participant regarding the viable CD34+ cells concentration, and a successful improvement effect of protocol standardization was also observed. However, we observed that better control over the critical parameters of the protocol did not have a significant effect on improving homogeneity in the enumeration of CD45+ cells.ConclusionsThe current practice in cord blood selection should now also consider relying on post-thaw CD34+ concentration, providing that all cord blood banks or outsourcing laboratories in charge of the analysis of post-thaw CB samples take into account the consensual recommendations provided in this work and adhere to a good-quality management system.     

Cytofluorometric methods for assessing absolute numbers of cell subsets in blood. European Working Group on Clinical Cell Analysis

The enumeration of absolute levels of cells and their subsets in clinical samples is of primary importance in human immunodeficiency virus (HIV)+ individuals (CD4+ T- lymphocyte enumeration), in patients who are candidates for autotransplantation (CD34+ hematopoietic progenitor cells), and in evaluating leukoreduced blood products (residual white blood cells). These measurements share a number of technical options, namely, single- or multiple-color cell staining and logical gating strategies. These can be accomplished using single- or dual-platform counting technologies employing cytometric methods. Dual-platform counting technologies couple the percentage of positive cell subsets obtained by cytometry and the absolute cell count obtained by automated hematology analyzers to derive the absolute value of such subsets. Despite having many conceptual and technical limitations, this approach is traditionally considered as the reference method for absolute cell count enumeration. As a result, the development of single-platform technologies has recently attracted attention with several different technical approaches now being readily available. These single-platform approaches have less sources of variability. A number of reports clearly demonstrate that they provide better coefficients of variation (CVs) in multicenter studies and a lower chance to generate aberrant results. These methods are therefore candidates for the new gold standard for absolute cell assessments. The currently available technical options are discussed in this review together with the results of some cross-comparative studies. Each analytical system has its own specific requirements as far as the dispensing precision steps are concerned. The importance of precision reverse pipetting is emphasized. Issues still under development include the establishment of the critical error ranges, which are different in each test setting, and the applicability of simplified low-cost techniques to be used in countries with limited resources.     

Cell analysis for hematopoietic stem/progenitor cell transplantation.

Cytometric analysis has become an important aspect in the quality control of cells in all phases of hematopoietic cell transplantation. In the stage of donor conditioning the counting of stem and progenitor cells is important and several reliable single platform tests for CD34+ cells have become available recently. It has been shown, that the count of certain subsets of CD34 may predict best time for harvesting stem cells better than just CD34. In many cases manipulation of the cell sample after collection from the donor is necessary before the cells are adequate for transplantation. Characterization of the resulting cell preparations requires reliable quantitative analysis of a variety of cell types like the enumeration of T-cells at the level of one in ten thousand for some allogeneic transplantations. It is discussed how these clinical requirements will need a refinement of cytometric procedures to achieve adequate clinical decisions.     

Flow cytometric enumeration of CD34+ hematopoietic stem and progenitor cells in leukapheresis product and bone marrow for clinical transplantation: a comparison of three methods

Flow cytometric enumeration of CD34+ hematopoietic stem and progenitor cells (HSCs) is widely used for evaluation of graft adequacy of peripheral blood and bone marrow stem cell grafts. In the present study, we review and compare the major counting techniques of stem and progenitor cells. The methods are: the Milan/Mullhouse protocol, two-platform ISHAGE (International Society of Hematotherapy and Graft Engineering) and single-platform ISHAGE analysis system. According to the Milan/Mulhouse protocol, HSCs are identified by CD34 antibody staining and easy gating strategy. The ISHAGE guidelines for detection of CD34+ cells are based on a four-parameter flow cytometry method (CD34PE/CD45PerCP staining, side and forward angle light scatter) thus employing multiparameter gating strategy. With two-platform ISHAGE protocol, an absolute CD34+ count is generated by incorporating the leukocyte count from an automated hematology analyser. The single-platform ISHAGE method to determine the absolute CD34+ count directly from a flow cytometer includes the use of Trucount tubes (Becton Dickinson) with a known number of fluorescent beads. CD34+ cells were quantified in mobilized peripheral blood, collected by leukapheresis, and bone marrow from 42 samples from patients with hematological malignancies. The differences against the means display low disagreement between the Milan/Mulhouse and ISHAGE protocols, with discrepancies of up to 2.5% (two-platform ISHAGE)--2.6% (single-platform ISHAGE) in enumeration of CD34+ cells in leukapheresis product and 4.8% (two-platform ISHAGE)--4.9% (single-platform ISHAGE) in bone marrow. Our results show high correlation among all three methods. Since the three protocols are compatible, choosing the most convenient in terms of costs, simplicity and compliance with clinical results appears to be a logical consequence.     

Enumeration of viable CD34(+) cells by flow cytometry in blood, bone marrow and cord blood: results of a study of the novel BD™ stem cell enumeration kit

Background aimsEnumeration of CD34⁺ cells in leukocyte-rich cell suspensions is important for clinical decision-making in stem cell transplantation. Single-platform flow cytometry assays offer the significant advantages of speed and reproducibility, and have therefore become the gold standard in stem cell enumeration. The clinical community has recently defined the need for stem cell enumeration kits that incorporate viability dyes. The purpose of this study was to evaluate a novel assay, BD Biosciences’ (BD) stem cell enumeration kit (SCE kit³), in relation to Beckman Coulter's (BC) commercially available BC Stem-Kit?.MethodsFresh/freeze-thawed samples from leukapheresis, bone marrow and cord blood, and fresh normal/mobilized blood, were analyzed with both assays (simultaneous detection of side/forward scatter and three fluorescence signals) on two flow cytometry platforms, BD FACSCanto II and BD FACSCalibur.ResultsResults from both assays were highly congruent, with an overall r² ≥ 0.99 (all specimen types included), a linear correlation across all CD34⁺ cell frequencies and concentrations, and an almost ideal steepness of the trend line.ConclusionsBoth assays functioned reliably. Being based on single-platform International Society of Hematotherapy and Graft Engineering (ISHAGE) guidelines and similar staining methods, both assays essentially come to identical results. For most specimen types, the viability of CD34⁺ cells was equal to overall leukocyte viability. In summary, in the hands of an experienced technician, the BD? SCE kit and the BC Stem-Kit are equivalent. The infrequent user might derive benefit from the fact that counting spheres are pre-pipetted into the Trucount tube for the SCE kit, making this assay less susceptible to pipetting inaccuracy.     

Human CD4+ T cells are predominantly distributed among six phenotypically and functionally distinct subsets

Human T cells are heterogeneous, varying in terms of their phenotype, functional capabilities, and history of Ag encounter. The derivation of a functionally relevant model for classifying CD4+ T cells has been hampered by limitations on the numbers of parameters that may be measured using classical four-color flow cytometry. In this study we have taken advantage of the introduction of reagents for five-color flow cytometry to develop a detailed, functionally meaningful scheme for classifying human CD4+ T cells. We show that CD4+ T cells are predominantly distributed among six of eight possible compartments, identified by the expression of CCR7, CD45RA, and CD28. We demonstrate novel phenotypic and functional correlates that justify the choice of these three molecules to define CD4+ T cell compartments. We note that CD4+ T cells with different Ag specificities are distributed differently among the six described subsets. On the basis of these results, we propose a cross-sectional model for classification of peripheral CD4+ T cells. Knowledge of where T cells lie on this model informs about their functional capacity and can reflect their history of Ag exposure.     

Simultaneous analysis of immunophenotype and apoptosis of murine thymocytes by single laser flow cytometry.

The study of the role of apoptosis in thymocyte development has been hampered by the lack of a means of directly immunophenotyping cells undergoing the early phase of apoptosis. This restriction has been overcome by single laser flow cytometry in which apoptosis is detected by Ethidium Bromide (EBr) staining and cell phenotype by binding of FITC-labelled antibody. The initial phase of apoptosis is observed as a cell population that stains faintly with EBr preceding the characteristically bright EBr-staining normally associated with cell death. Here we directly demonstrate using single laser flow cytometry that CD4+ CD8+ CD3low/CD3intermediate thymocytes undergo apoptosis in vitro in response to glucocorticoid treatment.     

Enumeration of CD34+ cells in cord blood: a variation on a single-platform flow cytometric method based on the ISHAGE gating strategy

Single-platform flow cytometric absolute cell counting protocols provide increased robustness for CD34+ cell enumeration by limiting potential sources of imprecision. However, samples with any cellular fragmentation or debris, such as cord blood samples, provide challenges for these assays. We describe a simple, robust absolute CD34+ cell counting protocol, suitable for cord blood, using TRUCOUNT absolute count tubes (BD Biosciences, San Jose, CA) and a modified ISHAGE (International Society for Hematotherapy and Graft Engineering) gating strategy. An advantage of TRUCOUNT tubes is that each tube is supplied with a known number of lyophilized fluorescent beads. The method includes no-wash fixative-free ammonium chloride red blood cell lysis and the viability dye, 7-amino actinomycin D, to exclude dead cells. The threshold was set on CD45 expression in the FL1 channel and an exclusion gate in the forward scatter channel reduced debris. No manual adjustment of the gating regions was required, even for samples in less than optimal condition. Comparison of the TRUCOUNT-ISHAGE protocol with the original dual-platform ISHAGE assay (n = 30) and the single-platform ISHAGE protocol using Flow-Count Fluorospheres (Beckman Coulter, Fullerton, CA; n = 22) showed high correlation (R(2) = 0.949 and 0.989, respectively) and no significant difference or bias for samples ranging from 22 to 600 CD34+ cells per microliter. Results are presented that demonstrate the detrimental effect of a fixative-containing lysis reagent when used in a lyse-and-wash procedure. The TRUCOUNT-ISHAGE protocol combines the attributes of TRUCOUNT tubes and the ISHAGE gating strategy to provide a single-platform protocol capable of achieving readily standardization of CD34+ cell enumeration.     

A false expression of CD8 antigens on CD4+ T cells in a routine flow cytometry analysis

The two-colour flow cytometry method applied in a routine enumeration of peripheral blood T lymphocyte subsets reveals that in some patients the entire population of CD4+ lymphocytes seems to express CD8 determinants as well. However, expression of the CD8 antigens on the cell surface is much lower in comparison with typical CD8+ cells. Moreover, in one-colour staining with an anti-CD8 antibody, cells with weak CD8 expression are not observed and only one typical population of CD8+ lymphocytes is seen. Investigating this phenomenon, we showed that after washing patient cells in RPMI before CD4/CD8 staining, the CD4+ T cell population did not show CD8 "co-expression". These results suggest that CD4+ lymphocytes, which seem to co-express CD8 antigen, in fact do not have this antigen on their surface. Moreover, after the addition of patient plasma to healthy donor cells prior to CD4/CD8 staining, a weak CD8 expression on normal CD4+ cells was noticed. Therefore we can assume that the agent(s) causing this phenomenon is/are present in the plasma of some patients. Altogether, these observations suggest that this phenomenon is nonspecific and probably results from cross-linking of anti-CD8 mAbs with anti-CD4 mAbs caused by factor(s) present in plasma of some patient. However, identification of that/these factor(s) requires further research.     

Development of an 8-color antibody panel for functional phenotyping of human CD8+ cytotoxic T cells from peripheral blood mononuclear cells

The study of CD8 positive cells in peripheral blood has become an essential part of research in the field of cancer immunotherapies, vaccine development, inflammation, autoimmune disease, etc. In this study, an 8-color flow cytometry panel, containing lineage and functional markers, was developed for the identification of CD8+ cytotoxic T cells in previously cryopreserved peripheral blood mononuclear cells from healthy human donors. By studying functional markers in naïve and CD3/CD28 activated T cells we demonstrate that the panel is capable of detecting protein markers corresponding to different T cell activation statuses. Data generated by flow cytometry were corroborated by different antibody based assay technologies to detect soluble cytokines. Our findings suggest that there is an inter donor variability in both baseline and activation responses. We have also successfully developed an antibody panel for flow cytometry that could be used to study cytotoxic function of CD8 T cells in clinical immunology research areas.     

Monitoring standards for molecular genetic testing in the United Kingdom, the Netherlands, and Ireland

Molecular genetic techniques have entered many areas of clinical practice. Public expectations from this technology are understandably high. To maintain confidence in this technology, laboratories must implement the highest standards of quality assurance (QA). External quality assessment (EQA) is recognized as an essential component of QA. The United Kingdom National External Quality Assessment Service (UKNEQAS) for Molecular Genetics, first set up in 1991, is currently the longest provider of EQA to molecular genetic testing laboratories in the UK, The Netherlands, and Ireland. Errors in the scheme are sporadic events. However, evidence from this and other EQA schemes suggests that a residual error rate persists, which should be taken into account in clinical practice. This EQA scheme has evolved from the respective scientific bodies of the constituent countries and retains a strong emphasis on collective peer review. It is essential that the steps taken to ensure quality in this rapidly expanding field are clear and transparent to participants and public alike. We describe the procedures developed and the governance imposed to monitor and improve analytical and reporting standards in participant laboratories and we compare our experiences with those of equivalent EQA services in the United States.     

临床检验前阶段室间质量评价方案的设计

在检验医学领域中,有很多研究已描述检验全过程不同阶段出现的最频繁的差错,且这些差错的很大部分都出现在检验前阶段。检验中阶段的误差登记然后反馈给参加者的方案已经由室间质评（External Quality Assessment,EQA）组织在大多数国家进行了数十年。但是迄今为止,只有很少的组织专注于检验前阶段,而且大多数的EQA组织并不提供检验前EQA方案（External Quality Assessment Schemes,EQAS）。执行和标准化检验前EQAS是比较困难的,认可机构也并没有对实验室参加这类方案提出要求。然而,一些正在进行的检验前阶段EQA计划是存在的,使用的方法可以分为三种类型：收集实验室检验前程序的信息、发放真实样品来收集可能影响测量程序的干扰因素、或者登记真实的实验室差错并将这些内容关联到质量指标。这三个类型有不同的侧重点和不同的实施挑战,这三者的结合可能是检出和监视出现在检验前阶段的广泛差错所必须的。     

Iron Oxide Nanoparticle Assisted Purification and Mass Spectrometry Based Proteolytic Mapping of Intact CD4+T Cells from Human Blood

Abstract

Iron oxide nanoparticles have been used for many years as clinical applications. We have developed a rapid immunoaffinity isolation method of CD4⁺T cells from a mixed cell population of human blood using iron oxide nanoparticles. Anti CD4-antibody has been attached to iron oxide nanoparticles after its surface modification. The antibody tagged iron oxide nanoparticle beads are simply incubated with the mixed cell population of human blood and CD4⁺T cells are purified using an external magnetic field. The purification level was checked by fluorescence microscopy and flow cytometry. The purified CD4⁺T cells were digested with trypsin with different time periods and the products were analyzed by MALDI-TOF mass spectrometry, without further fractionation or purification, to obtain its proteome pattern. A database search showed a number of peptide masses matched specific to T-cell peptide masses. These results indicate that iron oxide nanoparticles are useful for CD4⁺T cell purification, and mass spectrometry based proteolytic fingerprint is simple and swift for identifying putative surface biomarkers from the whole cell surfaces.     

Australasian CD34+ quality assurance program and rationale for the clinical utility of the single-platform method for CD34+ cell enumeration

BACKGROUND: Enumeration of CD34(+) cells should be accurate and comparable between institutions, particularly when making clinical decisions, evaluating data, and in clinical trials. An Australasian CD34(+) quality assurance program (QAP) has been established to compare CD34(+) cell results and method (Part 1). Unexpected variation in WBCCs led to Part 2 of this report. METHODS: Part 1: Methods reagents and results were evaluated for 12 QAP samples analyzed by 36-43 centers. Part 2: The effects of different anticoagulants on WBCC of 12 peripheral blood samples (PBs) were compared using three cell counters. To test the validity of applying the conclusions to clinical samples, the WBCCs of leukapheresed products and BM harvest were also compared. RESULTS: Part 1: In some samples, WBCCs determined by certain cell-counter groups were significantly different. Results for percentage of CD34(+) and CD34(+)/microL suggest that standardization on the lyse-no-wash and single platform (SP) method reduces variation of results between institutions. Part 2: Using different counters, PB WBCC in ACD-A showed greater variation than the same PB in EDTA. For PB in different anticoagulants, the extent of difference in WBCC for the same PB is dependent on the counter used. DISCUSSION: This CD34 QAP has identified ACD-A as an additional factor that contributes to the disparate WBCCs, which may further compromise the accuracy of CD34(+) cell counts obtained by the dual platform (DP) method, especially for leukapheresed products. In order to achieve greater accuracy within individual institutions, as well as permitting more reliable inter-institutional comparisons, our data supports the adoption of the SP as the standard method for CD34(+) cell enumeration.     

Impaired circulating myeloid DCs from myeloma patients

BACKGROUND: In clinical trials, cancer patients have received immunotherapy based on DCs generated from leukapheresed blood. It would therefore be an advantage to be able to measure blood levels and estimate the phenotype of DC before leukapheresis, to estimate the yield required for preparation of vaccines, or ex vivo stimulation of T cells for adoptive immunotherapy. METHODS: Recently, circulating lineage negative (Lin-) myeloid DC cells and their precursors have been identified by flow cytometry. We apply this strategy to the screening of blood samples from patients with multiple myeloma, in an attempt to characterize and quantitate the subset. By a direct flow cytometry approach, the blood levels of circulating lineage (CD3, CD19, CD14) negative, CD33++, HLA-DR+ cells were estimated before and following ex vivo cell differentiation, and phenotyped by MAbs with specificity against HLA-DR, HLA-ABC, CD1a, CD11c, CD33, CD40, CD49d, CD49e, CD54, CD80, CD83, and CD86. RESULTS: This study demonstrated that multiple myeloma patients have a 50% reduced blood level of Lin-, CD33++, HLA-DR+ myeloid DC, but a DC-precursor level within normal range. Furthermore, GM-CSF and IL-4 ex vivo stimulated DCs demonstrated an impaired up-regulation of the co-stimulatory molecule CD80 and the adhesion molecule CD54. DISCUSSION: These results may have clinical implications as a predictor for yield and functionality of the harvested DCs to be used in vaccination of myeloma patients.     

Large-scale protein interactome mapping: strategies and opportunities

Interactions between proteins are central to any cellular process, and mapping these into a protein network is informative both for the function of individual proteins and the functional organization of the cell as a whole. Many strategies have been developed that are up to this task, and the last 10 years have seen the high-throughput application of a number of those in large-scale, sometimes proteome-wide, interactome mapping efforts. Although initially the quality of the data produced in these screening campaigns has been questioned, quality standards and empirical validation schemes are now in place to ensure high-quality data generation. Through their integration with other 'omics' data, interactomics datasets have proven highly valuable towards applications in different areas of clinical importance.     

External Quality Assurance of Malaria Nucleic Acid Testing for Clinical Trials and Eradication Surveillance

Nucleic acid testing (NAT) for malaria parasites is an increasingly recommended diagnostic endpoint in clinical trials of vaccine and drug candidates and is also important in surveillance of malaria control and elimination efforts. A variety of reported NAT assays have been described, yet no formal external quality assurance (EQA) program provides validation for the assays in use. Here, we report results of an EQA exercise for malaria NAT assays. Among five centers conducting controlled human malaria infection trials, all centers achieved 100% specificity and demonstrated limits of detection consistent with each laboratory''s pre-stated expectations. Quantitative bias of reported results compared to expected results was generally <0.5 log₁₀ parasites/mL except for one laboratory where the EQA effort identified likely reasons for a general quantitative shift. The within-laboratory variation for all assays was low at <10% coefficient of variation across a range of parasite densities. Based on this study, we propose to create a Molecular Malaria Quality Assessment program that fulfills the need for EQA of malaria NAT assays worldwide.