Immunopathology / IMMUNOPHENOTYPE OF TRANSIENT STRESS LYMPHOCYTOSIS

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1 Immunopathology / IMMUNOPHENOTYPE OF TRANSIENT STRESS LYMPHOCYTOSIS Transient Stress Lymphocytosis An Immunophenotypic Characterization of the Most Common Cause of Newly Identified Adult Lymphocytosis in a Tertiary Hospital Nitin J. Karandikar, MD, PhD, Erin C. Hotchkiss, Robert W. McKenna, MD, and Steven H. Kroft, MD Key Words: Lymphocytosis; Naive; Memory; Effector; CD57 Abstract We prospectively evaluated 52 consecutive cases of newly identified absolute lymphocytosis to determine the hematologic and immunophenotypic features of transient stress lymphocytosis (TSL). The lymphocytosis in all cases was associated with an acute stressful event and ranged from 4,000 to 10,400/µL ( /L). Compared with healthy individuals, patients with TSL showed an increase in the total WBC, absolute lymphocyte (ALC), absolute neutrophil (ANC), and platelet counts with no difference in hemoglobin levels. Immunophenotypic analyses of 38 cases revealed increases in absolute numbers of T, B, and natural killer cells. Both CD4+ and CD8+ T cells were increased, predominantly accounted for by an increase in memory cell subsets, with no change in gamma/delta T cells. Follow-up studies showed a significant reduction in the ALC with a concurrent increase in the ANC and reduction in hemoglobin values. The reduction in lymphocytes at resolution was accompanied by reduction in all broad lymphocyte subsets. However, naive and memory subsets showed different patterns of alteration within the CD4+ and CD8+ populations, suggesting that acute stress differentially affects the in vivo distribution of these subsets. Absolute lymphocytosis is unusual in adults and, in many cases, may represent a lymphoproliferative disorder. Usually in lymphoproliferative disorders, the absolute lymphocytosis tends to be sustained over months or years. Transient absolute lymphocytosis is an underrecognized phenomenon that has been described in patients with emergency medical conditions, including trauma, cardiac conditions, sickle crises, abdominal pain, and obstetric emergencies. 1-4 This transient stress lymphocytosis (TSL) typically resolves within 24 to 48 hours of its diagnosis. 1 One previous report suggested that trauma- or stress-induced lymphocytosis may be an indicator of a relatively unfavorable clinical course. 3 Changes in circulating leukocyte and lymphocyte levels also have been described in conditions of psychologic and physical stress such as exercise. These are thought to be mediated, at least in part, by modulation of catecholamine and steroid hormones and cell adhesion molecules Exercise-induced lymphocytosis tends to resolve in a matter of a few hours but may show distinct temporal dynamics in different subsets of T cells. 11 Immunophenotypic analyses of TSL have revealed a global increase in B cells, T cells, and natural killer (NK) cells similar to responses seen following epinephrine administration. 2 However, detailed analysis of different subsets of lymphocytes in patients with stress lymphocytosis is still lacking, especially with regard to naive vs memory and/or effector T-cell subsets. Such analyses would elucidate the precise nature of this transient lymphocytic response and help shed further light on the dynamics of its regulation. We prospectively evaluated new cases of absolute lymphocytosis at an adult tertiary care and trauma center to determine the hematologic features of this process. We used Am J Clin Pathol 2002;117:

2 Karandikar et al / IMMUNOPHENOTYPE OF TRANSIENT STRESS LYMPHOCYTOSIS multiparameter flow cytometry for a detailed immunophenotypic evaluation of different subsets of circulating lymphocytes at diagnosis of the lymphocytosis and their dynamic shift during the resolution of this process. Materials and Methods Patients and Healthy Control Subjects Fifty-two consecutive adult patients from Parkland Memorial Hospital, Dallas, TX, with a newly identified absolute lymphocytosis (absolute lymphocyte count [ALC], 4,000/µL [ /L] or more) were accrued over a 22-day period. All patients with a known history of lymphocytosis were excluded from the study. Clinical history was obtained by chart review. Twelve age-matched healthy volunteers were recruited as control subjects. Follow-up data were obtained from available CBC counts for 20 of 52 patients. For 47 of 52 patients, either followup or immunophenotypic data were available. The other 5 cases showed characteristic clinical and hematologic findings of stress lymphocytosis and thus were retained in the analyses. Specimen Collection and Handling Blood was collected in EDTA anticoagulant. After obtaining CBC data and blood smears, the specimens were processed for flow cytometry. CBC Data and Peripheral Blood Smear Review The CBC data were obtained using a STAKS cell counter (Coulter, Miami, FL) in the Parkland Memorial Hospital clinical hematology laboratory. Blood smears were machine-stained with a Wright stain and reviewed by at least 2 hematopathologists (R.W.M., S.H.K., or N.J.K.) for lymphocyte morphologic features. Staining and Acquisition of Immunophenotypic Data The immunophenotype of the lymphocyte population was determined by using 4-color flow cytometry. A standard staining protocol was used, as described previously. 12 Following ammonium chloride RBC lysis, the WBC suspensions were stained with fluorochrome-tagged antibodies to a panel of lymphoid antigens. The following 6- tube panel was used (listed in the following order: fluorescein isothiocyanate/phycoerythrin/peridinin chlorophyll protein/allophycocyanin with clone names in parentheses): (1) IgG2a (X39)/IgG1 (X40)/IgG1/IgG1; (2) CD45RO (UCHL-1)/CD27 (L128)/CD3 (SK7)/CD4 (SK3); (3) T-cell receptor gamma-delta (MHGD01)/CD4/CD8 (SK1)/CD3; (4) CD57 (HNK-1)/CD56 (MY31)/CD8/CD3; (5) lambda/kappa/cd5 (L17F12)/CD19 (SJ25C1); and (6) CD10 (W8E7)/CD19/CD20 (L27)/CD38 (HB7). All antibodies were purchased from Becton Dickinson (BD)/Pharmingen, San Diego, CA, except lambda and kappa, which were purchased from Beckman Coulter, Miami, FL. The data were acquired on a BD FACSCalibur flow cytometer using BD Cellquest software. Analysis of Flow Cytometric Data The data were analyzed using BD Paint-A-Gate software. Controls Tube 1 in the antibody panel was the isotypic control tube used to determine the level of background staining (cutoff value or negative control), as described previously. 12 In addition, populations of cells in each specimen served as internal negative and positive controls. Memory vs Naive Cells Tube 2 was used to gate for T cells (CD3+) and divide them into CD4+ or CD4 populations. These were further separated into memory and naive cells based on CD27 and CD45RO expression Figure 1. (Naive T cells are CD27+, CD45RO. 13 ) CD4 CD3 CD27 CD45RO Figure 1 Enumeration of memory and naive CD4+ and CD8+ T lymphocytes. Tube 2 from the antibody panel was used to enumerate memory and naive subsets in each case. CD3+ lymphocyte-sized cells (total T cells) were further gated for CD4+ and CD4 populations (left panel). As confirmed from tube 3 (see the Materials and Methods section), the CD3+, CD4 subset was composed predominantly of CD8+ T cells (data not shown). CD27 and CD45RO staining was used to determine naive vs memory cells (right panel): CD27+, CD45RO cells were classified as naive (blue and black populations) and the rest as memory (orange and green). The gray populations represent non T cell events. In this patient, 13.32% of total events were CD4+ memory T cells (orange), 6.84% were CD4 memory cells (green), 5.29% were CD4+ naive cells (blue), and 3.52% were CD4 naive cells (black). 820 Am J Clin Pathol 2002;117:

3 Immunopathology / ORIGINAL ARTICLE Table 1 Blood Counts of Patients at Diagnosis Compared With Blood Counts of Healthy Control Subjects Patients (n = 52) Control Subjects (n = 12) Mean Range Mean Range P Absolute lymphocyte count, /µl ( 10 9 /L) 5,100 (5.1) 4,000-10,400 ( ) 1,800 (1.8) 1,200-3,400 ( ) <.0001 Absolute neutrophil count, /µl ( 10 9 /L) 6,200 (6.2) 2,100-19,300 ( ) 3,600 (3.6) 1,500-5,500 ( ).006 Total WBC count, /µl ( 10 9 /L) 12,400 (12.4) 6,300-26,500 ( ) 6,000 (6.0) 4,000-9,700 ( ) <.0001 Hemoglobin, g/dl (g/l) 13.1 (131) (63-164) 13.9 (139) ( ).09 Platelet count, 10 3 /µl ( 10 9 /L) 276 (276) (97-520) 221 (221) ( ).007 CD4/CD8 Ratio and gamma/delta T cells Tube 3 was used to determine the number of CD4- and CD8-bearing T cells, the CD4/CD8 ratio, and the CD3+, gamma/delta T-cell receptor bearing cells. Total T Cells and NK Cells Tube 4 was used to enumerate total T cells (CD3+) and total NK cells (all CD3, lymphocyte-sized cells that were CD56+ and/or CD57+ and/or CD8+). This tube also was used to enumerate the CD57+ T-cell subsets (CD8+ or CD8 ), which are thought to represent terminally differentiated effector T lymphocytes. B-Cell Analysis Tubes 5 and 6 were used to analyze the B-cell immunophenotype. Tube 5 determined the light chain distribution of mature B cells, and tube 6 was used to assay the CD20, CD10, and CD38 expression of the CD19+ B- lineage cells. Absolute Counts The absolute counts for each subset were determined by using the CBC data (WBC and ALC). Statistical Analysis A Student t test (unpaired) was performed to compare absolute numbers of different subsets of cells in patients vs healthy control subjects. A paired t test was used to compare counts in patients at diagnosis vs at resolution of lymphocytosis. P values of less than.05 were considered significant. Results Demographics and Clinical Manifestations Fifty-two consecutive patients with absolute lymphocytosis were evaluated as described in the Materials and Methods section. These comprised 22 male and 30 female patients with a mean age of 41.4 years (range, years). The 12 healthy control subjects were 5 men and 7 women with a mean age of 35.8 years (range, years). There were no statistical differences in the age and sex distribution in the 2 groups. All 52 patients presented clinically with an acute stressful event: 15 (29%) with trauma due to accidents (predominantly motor vehicle accidents), 7 (13%) with gynecologic and obstetric diagnoses, 6 (12%) with psychiatric emergencies, 5 (10%) with cardiac emergencies, 5 (10%) with thermal trauma, 4 (8%) with seizure disorders, 3 (6%) with sickle cell disease, 3 (6%) were postoperative, 2 (4%) with acute drug toxicity, 1 (2%) with acute infection, and 1 (2%) with a viral syndrome. CBC Counts and Morphologic Features The initial CBC data from the 52 patients and 12 healthy control subjects are shown in Table 1. There was a significant elevation of the total WBC count, which was due to an increase in both ALC and absolute neutrophil count (ANC). Absolute neutrophilia (ANC >7,000/µL [> /L]) was observed in 14 (27%) of 52 patients. In addition, there was a statistically significant increase in the platelet count with no difference in the hemoglobin values. Follow-up data were available for 20 patients and are shown in Table 2. The CBC values obtained at 7 to 569 hours showed a return of ALC to the reference range (median, 32 hours). At resolution of lymphocytosis, there was no significant change in the WBC count. Thus, the decreased ALC was replaced by a proportionate increase in the ANC. Absolute neutrophilia was present in 13 (65%) of 20 cases. A reduction in hemoglobin levels also was noted. Morphologic evaluation of blood smears revealed a characteristic spectrum of lymphocytes. The typical case showed increased numbers of small to medium-sized lymphocytes with a slightly decreased nuclear/cytoplasmic ratio and small cytoplasmic granules Image 1 (top left). A proportion of these cells showed an eccentric, often indented, nucleus with increased cytoplasmic basophilia. Only occasional large granular lymphocytes were present. Mild reactive features also were observed in other small and medium-sized lymphocytes in the form of cytoplasmic basophilia and plasmacytoid features (Image 1, top right). Am J Clin Pathol 2002;117:

4 Karandikar et al / IMMUNOPHENOTYPE OF TRANSIENT STRESS LYMPHOCYTOSIS Table 2 Blood Counts at Resolution Compared With Blood Counts at Diagnosis Diagnosis (n = 20) Resolution (n = 20) Mean Range Mean Range P Absolute lymphocyte count, /µl ( 10 9 /L) 5,500 (5.5 ) 4,000-10,400 ( ) 2,400 (2.4) 1,000-3,800 ( ) <.0001 Absolute neutrophil count, /µl ( 10 9 /L) 7,600 (7.6) 3,400-19,300 ( ) 10,600 (10.6) 3,900-31,200 ( ).048 Total WBC count, /µl ( 10 9 /L) 14,300 (14.3) 8,800-26,500 ( ) 14,000 (14.0) 6,800-33,600 ( ).838 Hemoglobin, g/dl (g/l) 12.6 (126) (63-160) 11.4 (114) (67-154).005 Platelet count, 10 3 /µl ( 10 9 /L) 290 (290) ( ) 248 (248) ( ).004 Image 1 Morphologic spectrum of transient stress lymphocytosis. Top left and right demonstrate cellular types seen in the typical case. Classic reactive lymphocytes (bottom left) or large granular lymphocytes (bottom right) were seen only in occasional cases and were not the characteristic feature. Downey II reactive lymphocytes (as seen in infectious mononucleosis) were not observed in the majority of cases. Only 3 (6%) of 52 cases demonstrated Downey III reactive lymphocytes (large cells with prominent nucleoli and deeply basophilic cytoplasm) along with increased large granular lymphocytes (Image 1, bottom left and right). Immunophenotypic Characteristics of Transient Stress Lymphocytosis at Diagnosis Immunophenotypic analyses were performed on 38 of 52 patients, as described in the Materials and Methods section. The absolute counts of different subsets of lymphocytes are presented in Figure 2 and compared with the values from healthy control subjects. There was a significant increase in total T cells, B cells, and NK cells. Both CD4+ and CD8+ T cells were increased with no change in the CD4/CD8 ratio. Within the CD4+ and CD8+ T-cell subsets, the elevation in counts was predominantly due to an increase in the memory T-cell subsets. Importantly, there was a significant increase in the CD57+ T-cell subsets; these are thought to represent terminally differentiated effector T cells and/or senescent T cells There was no difference in the total gamma/delta T-cell receptor bearing cells. There was no immunophenotypic evidence of a T- lineage lymphoproliferative disorder in any of the cases. There was no evidence of a B-lineage lymphoproliferative disorder in 37 (97%) of 38 cases. A single case revealed a lambda light chain excess (kappa/lambda = 0.38:1), suggesting a CD5, CD10 B-lineage clonal proliferation Figure 3. The absolute lymphocytosis in this case resolved within 59 hours, although the clonal process persisted. Hence, the stress-induced lymphocytosis served to unmask the lymphoproliferative disorder. Immunophenotypic Characteristics at Resolution Immunophenotypic analyses were performed on 11 follow-up specimens and are shown in Figure 4. At resolution, there was a significant decrease in the total numbers of T cells, B cells, and NK cells. Again, there was no significant change in the gamma/delta T cells. There was a significant reduction in both CD4+ and CD8+ T cells, especially in the CD4+ memory T-cell subset. The CD57+ T-cell subsets showed significant reduction at resolution. Discussion Transient absolute lymphocytosis is an underrecognized phenomenon that has been described in patients with emergency medical conditions including trauma, cardiac conditions, sickle crises, abdominal pain, and obstetric emergencies. 1-4 This TSL typically resolves within 24 to 48 hours of diagnosis. Some investigators also have reported similar changes and redistribution of circulating leukocyte subsets mediated by catecholamines. 5-9 These reports have shown predominant changes in the NK cell population associated with increased levels of circulating adhesion molecules such as intercellular adhesion molecule-1. Some investigators have reported a 822 Am J Clin Pathol 2002;117:

5 Immunopathology / ORIGINAL ARTICLE pan B- and T-cell lymphocytosis both in response to epinephrine administration and in some medical emergencies. 2,5 We performed a detailed immunophenotypic and hematologic analysis of different leukocyte subsets in patients with newly identified absolute lymphocytosis. All patients had an acute clinical picture. TSL was by far the most common cause of newly recognized absolute lymphocytosis. All patients with available follow-up data showed regression of absolute lymphocyte counts to the reference range within 7 to 569 hours (median, 32 hours). This median time frame is similar to that described in previous reports. 1-4 However, there were 2 patients who did not show resolution of lymphocytosis until 216 hours and 569 hours, respectively. In both cases, intermittent CBC data were available and showed sustained lymphocytosis. Thus, in individual cases, stress lymphocytosis may last well beyond 48 hours and still represent a transient nonneoplastic phenomenon. Only 1 patient showed evidence of a B-lineage lymphoproliferative disorder (Figure 3); the ALC in this case reached reference values after 59 hours. Thus, the stress-induced lymphocytosis helped reveal the underlying clonal disorder. The lymphocytes from these cases revealed characteristic, although not pathognomonic, morphologic features. Of note, in the vast majority of cases, the lymphocytes were distinct in morphologic appearance from those observed in classic viral syndromes. Similar to findings in previous studies, 2 the elevated ALC was associated with elevation in absolute numbers of all broad lymphocyte subsets, ie, T cells, B cells, and NK cells. Absolute numbers of CD4+ and CD8+ T cells also Mean Absolute Counts + 2 SEM ( 10 9 /L) A B C 4.0 P< P= P< P=.0005 P<.0001 P= P=.0001 P=.10 P=.002 P=.002 P= P=.007 P= Total T Cells Total B Cells Total NK Cells gamma/delta T Cells CD4+ T Cells CD8+ T Cells Figure 2 Immunophenotypic characteristics of transient stress lymphocytosis (TSL). Absolute counts of various immunophenotypic subsets of lymphocytes are shown. The black bars represent mean values from 38 patients with TSL at diagnosis of lymphocytosis, whereas the white bars represent 12 healthy individuals. The individual lymphocytic subsets are denoted on the x-axis, and the mean absolute counts + 2 SEM are indicated on the y-axis. NK, natural killer. were increased. However, there were notable differences within the finer subsets. The increase in CD4+ and CD8+ T cells was contributed predominantly by a marked elevation of memory T cells. Furthermore, there was a significant increase in the CD57+ T-cell subsets. Although the functional characteristics of this subset of T lymphocytes is not entirely clear, it seems to represent a population of terminally differentiated and/or senescent effector T cells. 13,17-20 It seems that, in response to stress, there is an immediate CD4/CD8 Ratio CD4+ Naive T Cells CD4+ Memory T Cells CD4 Naive T Cells CD4 Memory T Cells CD57+/CD8 T Cells CD57+/CD8+ T Cells CD19 kappa CD20 CD5 lambda CD10 Figure 3 Lambda light chain excess indicating clonal expansion of CD5, CD10 B-lineage cells. A single case showed evidence of clonal B-lineage proliferation. B, Excess of lambda light chain expressing B-lineage cells. The orange population represents lambda+ B-lineage cells (2.77% of all events), whereas the green events are kappa+ cells (1.06%). The black population represents total B- lineage cells, while the purple population denotes T lymphocytes. The CD5 (A), CD10 (C) immunophenotype suggests a marginal zone process. In this case, despite clonal proliferation, the absolute lymphocytosis resolved. Am J Clin Pathol 2002;117:

6 Karandikar et al / IMMUNOPHENOTYPE OF TRANSIENT STRESS LYMPHOCYTOSIS Mean Absolute Counts + 2 SEM ( 10 9 /L) Total T Cells P=.012 P=.017 P=.016 P=.075 Total B Cells Total NK Cells gamma/delta T Cells P=.007 P=.404 P=.034 CD4+ T Cells CD8+ T Cells P=.044 P=.004 P=.020 P=.040 P=.026 Figure 4 Immunophenotypic characteristics at resolution. Absolute counts of various immunophenotypic subsets of lymphocytes are shown. The black bars represent mean values from 11 patients with transient stress lymphocytosis at diagnosis of lymphocytosis, whereas the white bars represent the same patients at resolution of the lymphocytosis. The individual lymphocytic subsets are denoted on the x-axis, and the mean absolute counts + 2 SEM are indicated on the y-axis. NK, natural killer. P=.045 mobilization of this memory and/or effector population of T cells. This may represent a fundamental immunologic mechanism that enables the migration of these cells to sites of antigenic entry or presentation, aiding in an efficient anamnestic immune response. The typical acute immunologic response is thought to be neutrophilia. Of note, the elevated ALC in patients with TSL was associated with an elevated neutrophil count. At resolution of the lymphocytosis phase, there was a further proportionate increase in the ANC, with absolute neutrophilia in most patients. It is tempting to speculate that transient cytokine-mediated mobilization of lymphocytes (comprising cells of both the adaptive and innate immune systems) may be a primary phenomenon that accompanies the eventual mobilization of neutrophils, a first line of innate defenses. Resolution was defined as the return of the ALC to the reference range. However, at the level of the patient cohort, the ALC still remained significantly higher than that of healthy control subjects (P <.05), suggesting a gradual decrease in lymphocyte counts, which may take even longer to return to prediagnosis levels. There was a reduction in all broad lymphocyte subsets, including T cells, B cells, NK cells, and both CD4+ and CD8+ T cells. Again, the naive and memory subsets showed different degrees of alteration, with memory and effector populations showing the maximal reduction. Naive T cells also showed a reduction at resolution, although they were not significantly increased at diagnosis. These changes in CD4/CD8 Ratio CD4+ Naive T Cells CD4+ Memory T Cells CD4 Naive T Cells CD4 Memory T Cells CD57+/CD8 T Cells CD57+/CD8+ T Cells various lymphocytic subsets suggest an active redistribution of lymphocytes that is differentially affected by an acute stressful event. From the Division of Hematopathology and Immunology; Department of Pathology, University of Texas-Southwestern Medical Center at Dallas. Address reprint requests to Dr Karandikar: Dept of Pathology, 5323 Harry Hines Blvd, Dallas, TX References 1. Teggatz JR, Parkin J, Peterson L. Transient atypical lymphocytosis in patients with emergency medical conditions. Arch Pathol Lab Med. 1987;111: Groom DA, Kunkel LA, Brynes RK, et al. Transient stress lymphocytosis during crisis of sickle cell anemia and emergency trauma and medical conditions: an immunophenotyping study. Arch Pathol Lab Med. 1990;114: Pinkerton PH, McLellan BA, Quantz MC, et al. Acute lymphocytosis after trauma: early recognition of the high-risk patient? J Trauma. 1989;29: Thommasen HV, Boyko WJ, Montaner JS, et al. Absolute lymphocytosis associated with nonsurgical trauma. Am J Clin Pathol. 1986;86: Mills PJ, Berry CC, Dimsdale JE, et al. 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