Dendritic Cell Markers and PD-L1 are Expressed in Mediastinal Gray Zone Lymphoma.

AIMS: Mediastinal gray zone lymphoma (MGZL) is a rare entity with morphologic, immunophenotypic, and genetic features intermediate between classic Hodgkin lymphoma (CHL) and primary mediastinal large B-cell lymphoma (PMBL). It is challenging to differentiate from CHL and PMBL. A specific dendritic cell gene expression profile can distinguish CHL and MGZL from PMBL. We hypothesized that the dendritic markers fascin and CD123 may be helpful in distinguishing MGZL from CHL and PMBL. We also investigated programmed death-ligand 1 (PD-L1) expression in MGZL, which may have therapeutic significance in this difficulty to treat tumor. METHODS: Representative sections from 89 CHL, 20 PMBL, and 7 MGZL cases were stained for fascin, CD123, and PD-L1, and scored on a scale from 0 to 3+. Most (71%) MGZLs stained for CD123, as well as some (23%) CHLs, and few (11%) PMBLs. All MGZLs stained for fascin, as well as most (90%) CHLs, and approximately half (53%) of the PMBLs. PD-L1 was positive in all MGZLs, most (77%) CHLs and most (66%) PMBLs. CONCLUSIONS: Our study is the first to show CD123 is positive in a subset of formalin-fixed, paraffin-embedded MGZLs and CHLs, in contrast to PMBL which is largely negative. Staining for fascin was not significantly different between the lymphomas, but was less likely to be positive in PMBL. These findings suggest a role for CD123 and fascin in supporting diagnoses of MGZL and CHL, and in ruling out PMBL. By immunohistochemistry, PD-L1 is positive in MGZL, pointing to its therapeutic potential.

Human herpesvirus 6 lymphadenitis in drug rash with eosinophilia and systemic symptoms syndrome: a lymphoma mimic.

AIMS: Lymphadenopathy, haematological abnormalities and constitutional symptoms are among the non-specific manifestations seen in drug rash with eosinophilia and systemic symptoms (DRESS), an uncommon but potentially fatal cutaneous adverse drug reaction. The ubiquitous human herpesvirus 6 (HHV-6) plays a unique role in the pathogenesis of DRESS, with emerging data suggesting that reactivation occurs in most cases and contributes to the clinical manifestations, including lymphadenopathy. Further, in the appropriate clinical context, demonstration of HHV-6 reactivation may lend support to a diagnosis of DRESS. The histopathology of DRESS-associated HHV-6 lymphadenitis is reported rarely, with morphologic and immunophenotypic characteristics concerning for T cell lymphoma. The aim is to characterize the histopathology of HHV-6 lymphadenitis in the context of DRESS and to highlight this as an important cause of lymphadenopathy that may be a clinical, morphologic and immunophenotypic mimic of lymphoma. METHODS AND RESULTS: We describe a case of lymphoma-mimicking lymphadenitis in which the histopathological demonstration of reactivation of HHV-6 infection lent support to the clinical diagnosis of DRESS. CONCLUSION: Lymph node biopsies concerning for T cell lymphoma should be evaluated for HHV-6 involvement in a clinical context suggestive of DRESS.

Recurrent genetic defects in classical Hodgkin lymphoma cell lines.

Genetic analysis of classical Hodgkin lymphoma (cHL) has been hampered by the paucity of Hodgkin cells in biopsies and their poor growth in vitro. However, a wealth of information has been obtained from cHL cell lines. Here we report results of whole-exome sequencing and karyotypic analysis of five cHL cell lines. Four genes with potentially pathogenic single nucleotide variants (SNV) were detected in three cell lines. SNV were also detected in seventeen HL-related genes and three mitosis-related genes. Copy number variants were detected in four HL-related genes in all five cell lines. Given the high degree of aneuploidy in HL, mitosis-related genes were screened for defects. One mitotic gene (NCAPD2) was amplified in all five HL cell lines, and two genes (FAM190A, PLK4) were amplified in four cell lines. These results suggest that genomic instability of HL may be due to defects in genes involved in chromosome duplication and segregation.

Recommendations for gross examination and sampling of surgical specimens of the spleen.

This review examines handling and processing of spleen biopsies and splenectomy specimens with the aim of providing the pathologist with guidance in optimizing examination and diagnosis of splenic disorders. It also offers recommendations as to relevant reporting factors in gross examination, which may guide diagnostic workup. The role of splenic needle biopsies is discussed. The International Spleen Consortium is a group dedicated to promoting education and research on the anatomy, physiology, and pathology of the spleen. In keeping with these goals, we have undertaken to provide guidelines for gross examination, sectioning, and sampling of spleen tissue to optimize diagnosis (Burke). The pathology of the spleen may be complicated in routine practice due to a number of factors. Among these are lack of familiarity with lesions, complex histopathology, mimicry within several types of lesions, and overall rarity. To optimize diagnosis, appropriate handling and processing of splenic tissue are crucial. The importance of complete and accurate clinical history cannot be overstated. In many cases, significant clinical history such as previous lymphoproliferative disorders, hematologic disorders, trauma, etc, can provide important information to guide the evaluation of spleen specimens. Clinical information helps plan for appropriate processing of the spleen specimen. The pathologist should encourage surgical colleagues, who typically provide the specimens, to include as much clinical information as possible.

Human herpesvirus 6 positive Reed-Sternberg cells in nodular sclerosis Hodgkin lymphoma.

Classical Hodgkin lymphoma (HL) exhibits a bi-modal age distribution that suggests an infectious aetiology. However, most cases of nodular sclerosis HL (NSHL) are Epstein-Barr virus (EBV) negative (60-90%). Previous studies regarding human herpesvirus 6 (HHV-6) positivity of HL have led to conflicting results. In order to clarify this situation, we examined NSHL biopsies for the presence and distribution of HHV-6 by immunohistochemistry (IHC), polymerase chain reaction (PCR), and fluorescence in situ hybridization (FISH). PCR identified HHV-6 DNA in 86% of NSHL cases. As HHV-6 DNA was also identified in most cases of reactive lymphoid hyperplasia, we sought to localize the virus to specific cells by IHC, which detected HHV-6 in Reed-Sternberg (RS) cells of nearly half (48%) of NSHL cases. Dual CD30/HHV-6 immunostaining confirmed HHV-6 immunoreactivity in CD30+ RS cells, and HHV-6 PCR positivity was confirmed in laser capture microdissection-isolated CD30+ RS cells. FISH demonstrated multiple copies of HHV-6 genome in scattered cells. In contrast, EBV+ RS cells were identified in only 24% of the cases. HHV-6+ cases trended toward a younger age than EBV+ cases. These results conclusively demonstrate that RS cells in many cases of NSHL are HHV-6 positive, and suggest that HHV-6 may play a role in NSHL pathogenesis, particularly in younger patients with EBV-negative disease.

Detection of human herpesvirus DNA in Kikuchi-Fujimoto disease and reactive lymphoid hyperplasia.

Kikuchi-Fujimoto disease (KFD), or histiocytic necrotizing lymphadenitis, is a subacute inflammatory disorder most often seen in young women with clinicopathologic features suggestive of an infectious etiology. The most commonly suspected infectious agents in KFD are the human herpesviruses EBV, HHV6, HHV7 and HHV8. In order to identify herpesviruses in KFD, we have compared the frequency of detection of herpesvirus DNA with a recently developed real time PCR method, EBER in situ hybridization, and EBV latent membrane protein (LMP) immunostaining in 30 cases of KFD and 12 cases of reactive lymphoid hyperplasia (RLH). EBV DNA was commonly detected, while HSV2, CMV, HHV6, and HHV7 DNA were seldomly detected, and HSV1, VZV, and HHV8 DNA were not detected in KFD. EBV was also commonly detected in RLH. EBER-positive cells with apoptotic features were identified in necrotizing regions of many KFD cases, and LMP-positive cell debris was detected in one case. Viable EBER-positive cells were identified in four of twelve RLH cases, and rare LMP positivity detected in three cases. These data lend support to the notion that the necrotizing lesions in KFD may in some cases be due to a vigorous immune response to EBV-infected lymphoid cells.

Herpesvirus prevalence and viral load in healthy blood donors by quantitative real-time polymerase chain reaction.

BACKGROUND: After primary infection, human herpesviruses (HHVs) maintain long-term latent persistence, often punctuated years later by sporadic episodes of symptomatic lytic activation. Also, blood-borne herpesvirus from healthy persistently infected blood donors can lead to active primary infection of immunocompromised transfusion recipients. STUDY DESIGN AND METHODS: Utilizing a set of newly developed real-time polymerase chain reaction assays for detection and quantification of all eight human herpesviruses, the prevalence and viral DNA load of white cell-enriched blood from 100 randomly selected blood donors from the southeast Texas region are reported. RESULTS: Herpes simplex viruses 1 and 2 (HSV-1 and HSV-2), varicella-zoster virus (VZV), and HHV-8 DNA were not detected in any donor sample. In contrast, Epstein-Barr virus (EBV) (72%) and HHV-7 (65%) were commonly detected, HHV-6 (30%) was often detected (Type B only), and cytomegalovirus (CMV; 1%) was rarely detected. Median viral loads of positive samples (per milliliter of blood) ranged from 4278 for HHV-6 to less than 46 for EBV. CONCLUSIONS: These results suggest that the potential for transfusion-mediated transmission of herpesviruses from healthy adult blood donors is high for EBV and HHV-7; moderately high for HHV-6; uncommon for CMV; and rare for HSV-1, HSV-2, VZV, and HHV-8. Perhaps the most remarkable finding in this study was the detection of a single donor sample with greater than 6.1 x 10(7) HHV-6 Type B genome equivalents per mL blood. Given that this extraordinarily high level of HHV-6 DNA was obtained from a healthy adult blood donor, this phenomenon is likely unrelated to active infection or immunodeficiency.

Role of chromosome 1 pericentric heterochromatin (1q) in pathogenesis of myelodysplastic syndromes: report of 2 new cases.

Chromosome 1 pericentromeric heterochromatin (1q) has been shown to play an important role in the pathogenesis of non-Hodgkin lymphoma and multiple myeloma. Myelodysplastic syndrome (MDS) results from marrow failure in two or more cell lineages. Although trisomy 1q has been reported in MDS, it is usually present with additional common abnormalities such as trisomy 8, monosomy 5 or monosomy 7, leading to speculation that 1q abnormalities are mostly secondary events representing clonal evolution. We report two cases of MDS in which consistent involvement of 1q heterochromatin is seen as the primary clonal abnormality. Both patients presented with fatigue and pancytopenia. Based on the published reports and our cases, we propose that the 1q heterochromatin plays a vital role in the pathophysiology of MDS. Abnormalities involving 1q result in aberrant heterochromatin/euchromatin junctions, leading to gene dosage abnormalities. Further studies of 1q abnormalities in MDS might provide specific insights as to the exact role of the excess 1q heterochromatin in the etiology of MDS.

Comparative flow immunophenotypic features of the inflammatory infiltrates of Hodgkin lymphoma and lymphoid hyperplasia.

BACKGROUND: Hodgkin lymphoma (HL) is characterized by relatively few malignant Reed-Sternberg (RS) cells admixed within a reactive T cell rich inflammatory infiltrate. There is growing recognition that the HL-associated inflammatory milieu may enhance rather than inhibit growth of RS tumor cells. Since little is known of the immunophenotype of the HL inflammatory infiltrate we have performed a detailed retrospective comparison of the flow immunophenotype of HL and reactive lymphoid hyperplasia (RLH) to identify HL-specific immunophenotypic features. METHODS: Single cell suspensions from 59 lymph nodes involved by HL (at initial diagnosis) and 38 lymph nodes involved by RLH were subjected to a battery of 3-color combinations of well-characterized fluorochrome-conjugated monoclonal antibodies (DAKO) to a number of lymphocyte subsets. Cells were analyzed on a FACSCalibur flow cytometer with CellQuest software (Becton Dickinson). RESULTS: Overall, CD4+ T cells are increased and CD19+ B cells decreased in HL vs. RLH, yielding median T:B cell (CD3:CD20) ratios of 2.3:1 and 1.6:1, resp. There is no difference in median CD8+ T cell % (16% in HL & RLH). The T:B cell ratio is highest in nodular sclerosis (NSHL) and lymphocyte depletion (LDHL) subtypes, and lowest in mixed cellularity HL (MCHL). There is no significant difference in CD4:CD8 ratio in any comparison. NKT cells were slightly increased in HL vs. RLH, especially in MCHL. CD4+CD25+ regulatory T cells are significantly increased in HL (9%) vs. RLH (2%), especially in MCHL (29%) and NSHL (12%). EBV positivity in NSHL is associated with older age, decreased CD4+CD25+ regulatory T cells, CD4:CD8 ratio, and CD19/CD20+ B cells, and increased NKT cells, and CD14+ low forward-side scatter-gated monocytes. CONCLUSION: The cellular composition of the reactive lymphocytic infiltrate in HL differs significantly from that seen in RLH, with significant differences also noted between HL subtypes. In general, the HL infiltrate contains increased T cells (CD4+ and NKT subsets), decreased B cells, and increased regulatory T cells in comparison with RLH. The major difference between HL subtypes is decreased CD4+ T cells in MCHL as compared with NSHL and NLPHL. The most notable EBV-related difference in NSHL is increased regulatory T cells in EBV negative cases. While many differences in the reactive lymphocytic infiltrate of Hodgkin lymphoma and reactive lymphoid hyperplasia were identified, the sole difference that may prove to be of differential diagnostic value in flow cytometric analysis of HL versus RLH is the increased percentage of CD4+ bright CD25+ regulatory T cells in HL.

Prolonged preleukemic phase of chronic myelogenous leukemia.

We report the detailed features of a unique case of a 33-year-old male in whom a large percentage of marrow (68%) and blood (31%) cells carried the Philadelphia chromosome by cytogenetics and FISH, as well as the p210 BCR-ABL transcript by RT-PCR, for 15 months prior to development of chronic myelogenous leukemia. The patient was closely followed throughout the preleukemic period with repeat blood and bone marrow analysis with no hematologic or pathologic evidence of leukemia, despite harboring a large number (65%) of Ph+ marrow cells. we report the detailed history of a 33-year-old male who carried a large number of BCR-ABL-positive cells in the marrow and blood for more than 15 months before finally presenting with classic chronic myelogenous leukemia. This study is unique in that it provides the most detailed documentation of the hematologic, pathologic, and cytogenetic features of the preleukemic phase of chronic myelogenous leukemia ever reported.

Anatomical mapping of human herpesvirus reservoirs of infection.

Following primary infection, all eight human herpesviruses persist lifelong in the human host. However, a mapping of all anatomic sites of human herpesvirus persistence is lacking. Fresh tissue specimens representing approximately 40 major anatomic sites from eight autopsies were screened using a recently developed real-time PCR method for detection of all eight human herpesviruses. Patients with evidence of active herpesvirus infection (herpes simplex 1 (HSV-1), herpes simplex 2 (HSV-2), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), herpesvirus 6 (HHV-6), herpesvirus 7 (HHV-7), and herpesvirus 8 (HHV-8)) at the time of death were excluded to avoid detection of widely disseminated infection. Despite this precaution, widespread HSV-1 positivity (with blood positivity) was detected in one case-an elderly male who died of cardiac arrest. In a middle-aged male with HIV-AIDS, HSV-1 was found in neural and pharyngeal tissues, skin, cartilage, bone, and urinary bladder, whereas in two other cases, HSV-1 was restricted to neural tissues. HSV-2 was detected in a single site, the anus, in the male with HIV-AIDS. VZV was detected only twice, once in the adrenal gland and once in the small intestine. CMV was detected in three cases, most commonly in nasal mucosa, trachea, thyroid, intestine, and liver. EBV was detected in all eight cases, especially in nasal mucosa, tonsil, spleen, lymph node, tongue, and intestine, but in only two of six whole-blood specimens. HHV-6, like EBV, was detected in all eight cases, most commonly in salivary glands, thyroid, stomach, intestines, liver, and pancreas. HHV-7, like EBV and HHV-6, was detected in all eight cases, most commonly in salivary glands, tonsil, lymph nodes, and bone marrow. HHV-8 was detected in only two sites (both lymph nodes) from two cases. Herpesviruses were detected in three of six whole-blood specimens, including HSV-1, EBV, HHV-6, and HHV-7. These results represent the most comprehensive mapping of herpesvirus tissue distribution in humans reported to date.

Regulation of adrenal glucocorticoid synthesis by interleukin-10: a preponderance of IL-10 receptor in the adrenal zona fasciculata.

Several lines of evidence indicate that cytokines can affect adrenal function. To date most of these cytokines have been shown to be pro-inflammatory, such as interleukin (IL)-1, tumor necrosis factor (TNFalpha), and IL-6. However, we have previously shown that IL-10-/- (IL-10 knockout) mice have higher serum corticosterone levels than IL-10+/+ (wild type) mice following acute immune and physiologic stress, implying that IL-10, an anti-inflammatory cytokine, regulates glucocorticoid synthesis in a negative manner. Here, we show that IL-10 knockout mice produce more corticosterone under basal conditions as well (shown by ELISA). We further support this contention by showing that in Y-1 adrenocortical cells IL-10 inhibits steroid production (StAR) (measured by the production of the corticosterone precursor, progesterone), the expression of steroidogenic acute regulatory protein (semi-quantitative RT-PCR), as well as the activity of the proximal steroidogenic enzymes P450scc and/or 3beta-hydroxysteroid dehydrogenase (3beta-HSD) (measured by progesterone production in 22(R)-hydroxycholesterol-treated cells). Interestingly, all of the above-mentioned effects of IL-10 occur through its inhibition of ACTH effects, but not by IL-10 alone. Furthermore, immunocytochemistry data shows that the region of the adrenal gland responsible for the vast majority of corticosterone synthesis, the zona fasciculata, predominantly expresses the IL-10 receptor 1 (IL-10R1), with little expression in the zona glomerulosa and reticularis. These data demonstrate that IL-10 could play an important role in the regulation of glucocorticoid biosynthesis and in maintenance of homeostasis and immunity during periods of stress.

Distribution and phenotype of Epstein-Barr virus-infected cells in human pharyngeal tonsils.

Although Epstein-Barr virus (EBV) is often found in human tonsils, it remains to be precisely determined in what cells and microenvironment the virus is present. Although generally regarded as a B lymphotropic virus, EBV is associated with non-B-cell tumors, for example, NK/T-cell lymphoma, carcinoma, and leiomyosarcoma. To provide a basis for understanding the origin and biology of EBV-infected non-B cells, the immunophenotype of all EBV-infected cells in reactive human tonsils was determined by subjecting tonsil sections to dual/triple EBER in situ hybridization and immunohistochemistry with monoclonal antibodies to T cells (CD3, CD4, CD8, CCR3), B cells (CD20), plasma cells (CD138), natural killer (NK) cells (PEN5), and epithelial cells (cytokeratin), as well as frozen section immunostaining with antibodies to EBV latent proteins EBNA1, EBNA2, LMP1, and EBV early protein BZLF1. Most tonsils contained nearly equal numbers of EBNA1- and LMP1-positive cells (latency program) while only a few contained EBNA2-positive cells (growth program). More than 1000 EBER-positive cells from six tonsils were detected in the interfollicular zone (59%), tonsillar crypts (26%), and follicles (15%). Most (82%) EBER-positive cells are CD20-positive B cells, 7% are CD3-positive T cells, and 11% are cells of indeterminate lineage, often with plasmacytoid morphology. However, no EBER-positive plasma cells were identified. Rare EBER-positive NK cells and EBER/BZLF1-positive epithelial cells were identified. The direct demonstration of EBV within rare T cells, NK cells, and epithelial cells in reactive human tonsils provide a basis for further understanding of the origin of EBV-associated tumors of non-B-cell type.

Comprehensive analysis of genetic polymorphisms in the interleukin-10 promoter: implications for immune regulation in specific ethnic populations.

The association of interleukin-10 (IL-10) promoter single-nucleotide polymorphisms (SNPs) as risk factors for certain inflammatory diseases, viral infections, cancers, and transplant rejection have been the subject of recent studies. The SNPs -1082 G --> A, -819 C --> T, and -592 C --> A, which have been associated with differential IL-10 production, are strongly linked with ethnicity. In this study, we determined the ethnic distribution of IL-10 promoter SNPs and their haplotype rates among Hispanics, African Americans, and Caucasians from Texas and Ashkenazi Jews from New York. Significant differences in prevalence rates of IL-10 SNPs (and their haplotype distribution) were found. African Americans and Hispanics have a lower rate of putative high-producer SNPs and a higher rate of low IL-10 producers when compared to Caucasians or Ashkenazi Jews. No statistically significant differences in allelic frequencies and haplotype rates were observed between Caucasians and Ashkenazi Jews. This study provides critical new information on the ethnic distribution of IL-10 promoter SNPs in a regional U. S. population and is the first to analyze the rate of SNPs in an unstudied ethnic population, Ashkenazi Jews. Knowledge of IL-10 promoter polymorphisms may prove useful in prediction of immunization responses, disease severity, and in the intelligent design of customized immunotherapy.

Nevus-cell aggregates in lymph nodes: fine-needle aspiration cytologic findings and resulting diagnostic difficulties.

We report a case of nodal nevus present in enlarged lymph nodes with changes of dermatopathic lymphadenopathy sampled by fine-needle aspiration (FNA) cytology prior to clinical evaluation of the patient. This lymph node pathology was established later by lymph node excisional biopsy, by which along with a skin biopsy the dermatopathic lymphadenopathy was tentatively attributed to early mycosis fungoides. The FNA revealed fairly atypical melanotic tissue from the dermatopathic lymphadenopathy along with nodules of uniform melanocytic nevoid cells, the presence of which in combination with the dermatopathic atypical tissue provided a tentative diagnosis of metastatic melanoma of unknown primary, with the diagnosis of nodal nevus presented as a less likely possibility. This is to our knowledge the first cytologic report on FNA of nodal nevus, which besides presenting cytologic findings of this entity highlights some of the problems related to providing an accurate diagnosis, if this exceptionally unusual pathologic entity is encountered in lymph nodes sampled for enlargement from pathologies unrelated to this entity. The subject of nevus changes in lymph nodes is briefly discussed.

Species identification of all eight human herpesviruses with a single nested PCR assay.

There are eight currently known human herpesviruses, all of which are capable of latent persistence and reactivation following primary infection. Herpesvirus induced disease is common, widespread, and associated with significant morbidity, particularly in the immunocompromised human host. Current methods of herpesvirus detection include viral culture and polymerase chain reaction (PCR). A robust PCR method based upon amplification of the highly conserved herpesvirus DNA polymerase gene that is capable of detection of all eight human herpesviruses, including EBV and HHV-6 subtypes, from clinical material is described. Species identification of PCR products is accomplished by either of two methods, chemiluminescent dot blot hybridization and heteroduplex mobility shift assay, both of which allow for simultaneous detection of multiple herpesviruses. This method should prove useful for rapid and accurate species identification of all eight human herpesviruses from clinical material.

Comparative immunophenotypic features of EBV-positive and EBV-negative atypical lymphocytosis.

BACKGROUND: Atypical lymphocytosis is a common peripheral blood abnormality seen not only in Epstein-Barr virus (EBV)-associated acute infectious mononucleosis but also in other conditions, including other viral infections, cancer, immune reactions, etc. Despite numerous reports of individual immunophenotypic alterations in EBV-positive infectious mononucleosis, a detailed comparative analysis of the immunophenotypic changes of peripheral blood lymphocyte subsets in infectious mononucleosis and other forms of atypical lymphocytosis is lacking. METHODS: Multiparametric flow immunocytometry with 26 monoclonal antibodies was performed on peripheral blood lymphocytes from 97 cases of atypical lymphocytosis and 37 normal controls. Atypical lymphocytosis was defined as absolute lymphocytosis with >10% atypical lymphocytes. Absolute or relative mean values of various lymphocyte subsets from EBV-positive cases, EBV-negative cases, and normal controls were compared with the Student's t-test. RESULTS: Highly significant abnormalities detected in atypical lymphocytosis include increases in CD3+/CD8+, CD3-/CD16/56+, CD3+/gammadelta+, CD8+/CD48-, CD8+/CD57-, CD8+/CD95+, CD4+/CCR5+ CD4+/CD7-, CD4+/CD43-, CD4+/CD48-, and CD4+/CD62L- subsets. In contrast, no change in absolute CD4+ T cell and CD19+ B cell counts is seen. When compared with EBV-negative cases, EBV-positive cases are characterized by younger age, and increased numbers of absolute lymphocytes, atypical lymphocytes, CD8+ cells, NK cells, gammadelta T cells, CD8+/CD45RO+ cells, CD8+/CD57- cells, and CD8+/CD28+ cells. CONCLUSIONS: All forms of atypical lymphocytosis are characterized by a marked increase in activated CD8-positive T cells, a moderate increase in NK cells, and no increase in CD4-positive T cells and B cells. Although morphologically indistinguishable, EBV-associated mononucleosis is characterized by several significant differences in peripheral blood lymphocyte subsets when compared with EBV-negative atypical lymphocytosis, most notably increased numbers of CD57-negative CD8 T cells and gammadelta T cells.