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Background: Manual microscopy is the current reference method for white blood cell (WBC) differential counts. However, manual counts are time and labor intensive, difficult in patients with low WBC counts, and can misclassify cells having difficult morphology. We investigated an 8-color, single-tube, lyse no-wash flow cytometric method to perform an extended 8-part differential as a potential replacement reference method for WBC differential enumeration.Methods: Whole blood was stained using a panel of antibodies including CD45APC-Cy7, CD161CD19FITC, CD331CD64PE-Cy5, CD123PE, HLA-DRPE-Cy7, CD341CD117APC, and CD38A594 with the membrane permeant DNA binding dye Hoechst 34580 to generate an 8-part differential (lymphocytes, granulocytes, monocytes, eosinophils, basophils, immature granulocytes, blasts, and nucleated RBCs) using TruCount beads to generate absolute counts for all populations. Manual and instrument differentials were generated for 300 blood samples ranging from normal to complex. Results were compared with the flow cytometric differential.Results: The flow cytometric WBC and differential correlated well with the Sysmex XE2100 hematology analyzer and gave comparable results to the manual differential. Areas of greatest discordance included enumeration of populations present at low numbers and misclassification of cells with unusual morphology by the manual method. This study describes a novel single-tube flow cytometric method for performing a WBC count and 8-part differential that performs well with both normal and difficult patient samples. These findings confirm the results of prior studies supporting the use of a flow cytometric differential as an improved reference method for the WBC differential and extend prior efforts by allowing positive identification of most cell populations.

eThe presence of inherited chromosomally integrated human herpesvirus 6 (ciHHV-6) in hematopoietic cell transplant (HCT) donors or recipients confounds molecular testing for HHV-6 reactivation, which occurs in 30 to 50% of transplants. Here we describe a multiplex droplet digital PCR clinical diagnostic assay that concurrently distinguishes between HHV-6 species (A or B) and identifies inherited ciHHV-6. By applying this assay to recipient post-HCT plasma and serum samples, we demonstrated reactivation of HHV-6B in 25% (4/16 recipients) of HCT recipients with donor-or recipient-derived inherited ciHHV-6A, underscoring the need for diagnostic testing for HHV-6 infection even in the presence of ciHHV-6. Human herpesvirus 6A and -B (HHV-6A and -B) are known to be able to integrate within human chromosomal subtelomeric regions through homologous recombination at HHV-6 direct repeat regions (1). If this integration occurs within germ line cells, the offspring produced from those cells carry a copy of HHV-6A or -B in every nucleated cell; this condition is called inherited chromosomally integrated HHV-6A or -B (ciHHV-6A or -B) (2). The presence of ciHHV-6A or -B in every cell of the body has unknown effects (3), but two recent studies suggested a link between inherited ciHHV-6A or -B and angina pectoris (4, 5). Also, several reports have demonstrated HHV-6 reactivation and associated disease from inherited ciHHV-6 (6, 7).HHV-6B reactivation (but not HHV-6A reactivation) occurs in 30 to 50% of hematopoietic cell transplant (HCT) recipients, typically within the first 2 to 6 weeks posttransplantation. This has been associated with complications, such as central nervous system dysfunction, fever and rash, myelosuppression, graft rejection, and acute graft-versus-host disease (GVHD), some of which have also been reported after solid organ transplantation (8). While further research is needed to determine the clinical significance of inherited ciHHV-6A or -B after transplantation, it is well recognized that inherited ciHHV-6A or -B complicates molecular diagnostic testing for HHV-6B reactivation (9, 10). Specifically, tissue and blood samples from patients with inherited ciHHV-6A or -B will have high levels of HHV-6A or -B detected by routine quantitative PCR (qPCR), obscuring detection of HHV-6B reactivation.To aid in the recognition of inherited ciHHV-6 in the transplant setting, we recently developed a clinical droplet digital PCR (ddPCR) assay to identify inherited ciHHV-6 in cellular patient specimens (11). A subsequent study demonstrated the utility of ddPCR in distinguishing HHV-6A and -B (12). Here we describe an improved assay that identifies inherited ciHHV-6 and determines which species, HHV-6A or HHV-6B, is responsible for the integration by using a single reaction mixture. We also show that this assay can aid in the diagnosis of reactivated HHV-6B in HCT recipients with inherited ciHHV-6A, which further highlights the need for improved diagnostics for HHV-6 reactivation, particularly in immunosuppressed patien...

Human herpesvirus 6 (HHV-6) is an important cause of meningitis and meningoencephalitis. As testing for HHV-6 in cerebrospinal fluid (CSF) is more readily available using the FilmArray Meningitis/Encephalitis panel (FA-ME; BioFire Diagnostics, Salt Lake City, UT), we aimed to determine the clinical significance of detecting HHV-6 in order to identify true infections and to ensure appropriate antiviral initiation. Chart review on 25 patients positive for HHV-6 by FA-ME was performed to determine clinical presentation, comorbidity, treatment, and outcome. The presence of chromosomally integrated HHV-6 (ciHHV-6) DNA was also investigated. Of 1,005 children tested by FA-ME, HHV-6 was detected in 25 (2.5%). Five patients were diagnosed with either HHV-6 meningitis or meningoencephalitis based on HHV-6 detection in CSF, clinical presentation, and radiographic findings. Detection of HHV-6 by FA-ME led to discontinuation of acyclovir within 12.0 h in all 12 patients empirically treated with acyclovir. Six of the 12 patients were started on ganciclovir therapy within 6.8 h; 4 of these were treated specifically for HHV-6 infection, whereas therapy was discontinued in the remaining 2 patients. CSF parameters were not generally predictive of HHV-6 positivity. The presence of ciHHV-6 was confirmed in 3 of 18 patients who could be tested. Five of the 25 patients included in the study were diagnosed with HHV-6 meningitis/meningoencephalitis. FA-ME results led to discontinuation of empirical antiviral treatment in 12 patients and appropriate initiation of ganciclovir in 4 patients. In our institution, detection of HHV-6 using FA-ME led to faster establishment of disease etiology and optimization of antimicrobial therapy.

2547 The flow cytometric detection of minimal residual disease (MRD) relies on the differential expression of antigens between normal and leukemic cell populations of similar lineage. In T-cell lymphoblastic leukemia (T-ALL), the principal normal populations from which leukemic blasts must be distinguished are mature T cells and NK cells, as immature T cells are not normally present in peripheral blood or bone marrow. Current methods rely on a relatively small number of antigens, some of which are not stable following therapy. In particular, immature antigens such as TdT and CD99 that distinguish leukemic and mature cells at diagnosis often revert to mature levels after therapy. The subset of T-ALL cases having expression of surface CD3 can be particularly problematic. Consequently, the identification of novel antigens to supplement those currently in use is highly desirable. We undertook a systematic search for novel antigens capable of distinguishing T-ALL from mature T cell and NK cell populations using a high-throughput flow cytometric screening technique. LyoPlates (Becton-Dickinson) containing antibodies against 242 unique antigenic specificities in a 96 well plate format were used to assay mononuclear cells obtained from 3 normal peripheral blood donors and 9 pediatric patients with T-ALL. The T-ALL cases covered the range of immunophenotypes seen in this disorder. The 9-color assay (1 detection and 8 gating fluorochromes) was performed on an LSRII and was capable of identifying discrete T cell, NK cell and Blast cell subpopulations. Comparison of the median fluorescence intensity of each of the 242 unique antigens identified CD48 as one of the few antigens that showed consistent differential expression between mature T cells and NK cells in comparison to leukemic blasts from T-ALL. To determine whether differential expression of CD48 represents a universal characteristic of the disorder, we assayed 126 consecutive pretreatment T-ALL samples from pediatric patients enrolled on Children's Oncology Group protocol AALL0434. An 8 color flow cytometric assay was employed using the following antibody combination: CD48 FITC, CD5 PE, CD16+CD56 PE-Cy5, CD3 PE-Cy7, CD8 V450 or BV421, CD4 A594, CD7 APC, CD45 APC-Cy7. Comparison of the median fluorescence intensity of CD48 between leukemic blasts and normal T and NK cells within the same specimen revealed consistently decreased expression of CD48 on leukemic blasts (see Figure 1) with some variation in intensity. This finding was highly statistically significant (p<4E-105). The stability of CD48 expression following therapy was determined by assay of CD48 expression in 126 bone marrow samples obtained at day 29 following induction chemotherapy, representing paired samples from the patients whose pretreatment samples were assayed above. The same 8 color reagent combination was used in conjunction with the standard MRD assayed utilized for the treatment protocol, a two-tube 9 color assay. Of these samples, 50 (39.7%) showed detectable MRD using the standard assay. At end induction, the ratio of CD48 median fluorescence intensity between the leukemic MRD population and normal T and NK cells within the same sample remained abnormal in most cases. It was unchanged in 26% of cases, decreased (i.e. became more aberrant) in 24%, and increased in 50%, but in most of the latter cases remained significantly <1. However, in 5 cases (10%), CD48 increased to the level seen on normal mature T and NK cells. The changes following therapy were due largely to changes in the expression of CD48 on leukemic blasts as the level of expression of normal T and NK cells was more stable. No association between apparent immunophenotypic maturational stage or surface CD3 expression was identified. We conclude that the expression of CD48 is consistently reduced on leukemic blasts from patients with pediatric T-ALL in comparison to normal mature T and NK cells at the time of diagnosis. Following therapy, the expression of CD48 undergoes modulation, but remains different from mature T and NK cells in 90% of patients. This suggests that CD48 is a useful addition to reagent combinations for the purpose of MRD assessment in pediatric T-ALL. Disclosures: Wood: BD Biosciences: Research Funding. Borowitz:BD Biosciences: Research Funding.