Pediatric and Developmental Pathology 17, 112–117, 2014 DOI: 10.2350/13-08-1321-OA.1 ª 2014 Society for Pediatric Pathology
Monocytopenia as a Diagnostic Clue to Pediatric B-Lymphoblastic Leukemia with Rare Circulating Blasts SUNITA I. PARK*
AND
BEVERLY B. ROGERS
Children’s Healthcare of Atlanta, 1405 Clifton Road, NE, Egleston Children’s Hospital, Department of Pathology, First Floor, Tower One, Atlanta, GA 30322, USA
Received April 12, 2013; accepted June 27, 2013; published online July 1, 2013.
ABSTRACT
INTRODUCTION
B-lymphoblastic leukemia/lymphoma (B-LL) is the most common childhood cancer. Circulating blasts in the peripheral blood may be rare (1%) and missed, even when flow cytometric immunophenotyping is performed, leading to a false-negative report. The records from all patients with a new diagnosis of B-LL between January 2009 and December 2011 at our institution were reviewed. Of 130 cases with peripheral blood flow cytometry, 15 had a blast count of 1%, with 14 having electronic files for gating monocytes. The percentage of monocytes by flow cytometry and absolute monocyte counts (AMCs) were compared with peripheral blood samples that were negative by flow cytometry, sent due to cytopenia of at least 1 lineage (n 5 39). The monocytes from the patients with leukemia averaged 0.8% and were statistically fewer than the negative controls, which averaged 7.1% (P , 0.001). Eleven of the 14 (79%) patients with leukemia had monocytes ,1%, compared to only 3 (8%) of the negative controls. The AMCs were also significantly lower (P , 0.001), with 93% of the leukemia group having an AMC ,100 cells/mL, compared to only 28% of the negative controls. In patients with cytopenias, percentage of monocytes may be an important diagnostic clue in determining the presence of occult leukemia. If flow cytometry is performed, acquisition of more than the standard 10 000 events is necessary to adequately assess for leukemia. If monocytes are ,1% by flow cytometry in the setting of cytopenias, bone marrow examination is recommended, even with negative peripheral blood flow cytometry.
B-lymphoblastic leukemia/lymphoma (B-LL) is the most common childhood tumor, with an estimated 3000 new cases annually in the United States [1]. The majority of patients present with cytopenias, and blasts are easily seen on peripheral blood smear. Flow cytometric immunophenotyping of the peripheral blood is then performed to characterize the immunophenotype of the blasts, which often express dimmer CD45 than mature B cells and may aberrantly express CD10, CD34, CD58, and terminal deoxynucleotidyl transferase, with lack of surface kappa or lambda staining [1]. Given the expected absence of normal B-cell progenitors (hematogones) in the peripheral blood, which may have a similar phenotype, the leukemic blasts are typically easy to identify by flow cytometry. Rarely, patients present with a very low circulating blast burden, which may be 1%, making the diagnosis challenging, even with peripheral blood flow cytometry. In a paper describing a rare event analysis, Allan and Keeney [2] showed that, given a coefficient of variation of 20% in detecting a positive population, 25 events will define an abnormal cluster of cells. If a sensitivity of 0.01% is desired, 2.5 3 105 cells need to be acquired [2], which is much greater than the standard 10 000 events/ tube [3] that is used in many flow labs. This level of sensitivity may be challenging to achieve in hypocellular samples, samples with low specimen volume, or samples with debris and/or platelets, which can fall into the CD45 dim region and obscure a minor abnormal population. Failing to detect the B lymphoblasts will lead to a falsenegative flow report, which may lead to a false sense of security and/or delay in examining the bone marrow. Monocytes are myeloid-derived white blood cells that aid in antigen presentation. They circulate briefly in the peripheral blood and then migrate to the tissues, where they mature into various cells of the monocyte/histiocyte/ immune accessory cell system. Monocytes are increased in physiologic conditions, such as during the neonatal period and during marrow recovery from agranulocytosis. They are also increased in reactive conditions, such as
Key words: B-lymphoblastic leukemia, flow cytometry, monocytopenia, pediatric
*Corresponding author, e-mail: [email protected]
Table 1.
Monoclonal antibodies used for immunophenotypic characterization
Monoclonal antibody
Clone
Used for
Source
CD19 CD10 CD34 CD20 CD58 TDT Kappa/lambda CD33 CD64
SJ25C1 HI10a 8G12 L27 1C3 (AICD58.6) HT1, HT4, HT8, HT9 TB28-2/1-155-2 P67.6 22
B-LL B-LL B-LL B-LL B-LL B-LL B-LL Monocytes Monocytes
BD BD BD BD BD Pharmingen Beckman Coulter Dako BD Beckman Coulter
B-LL indicates B-lymphoblastic leukemia; BD, Becton Dickinson; TDT, terminal deoxynucleotidyl transferase.
infection, autoimmune diseases, and certain neoplasms [4]. Decreased monocytes are uncommon, and the differential diagnosis includes marrow failure states, such as aplastic anemia, and glucocorticoid administration, hemodialysis, sepsis, and certain hematologic malignancies, such as hairy cell leukemia [5]. This study examines the role of using decreased monocytes, as determined either by flow cytometry or absolute monocyte count (AMC), as a diagnostic clue to aid in the diagnosis of B-LL with very low circulating blasts.
METHODS Study groups With appropriate institutional review board approval, the records from all patients with a new diagnosis of B-LL at Children’s Healthcare of Atlanta (CHOA) during a 3-year period (January 2009–December 2011) were retrospectively reviewed. Of 171 cases, 130 had peripheral blood flow cytometry performed at our institution.
B-LL with 1% blasts Fifteen of the aforementioned 130 patients had a blast count ranging from 0.08%–1.0% by flow cytometry. In all cases, a bone marrow biopsy confirmed the diagnosis of B-LL by both morphology and flow cytometry. The percentage of monocytes, as determined by flow cytometry and AMC, was compared to that of the negative control group. Fourteen of the 15 cases had electronic files that allowed for re-gating of the monocytes by CD33 and CD64 (see following).
Controls This group was made up of all patients seen in 2011 whose peripheral blood was sent for flow cytometry because of cytopenia of at least 1 lineage stated in the clinical history. Of 43 total cases, 39 were included in the study, as they had electronic files that allowed gating of the monocytes. Patients with a prior diagnosis of a hematologic malignancy, solid tumor, or Down syndrome were excluded. Clinical follow-up and bone marrow examination, when performed, were negative for leukemia.
B-LL with .25% blasts Flow cytometry reports from 52 patients with ‘‘typical’’ B-LL who presented with blast counts .25% in the peripheral blood were reviewed. The percent monocytes from the flow cytometry report was recorded, which was generated by gating CD14+ cells with low to intermediate side scatter properties.
Comparison with T-lymphoblastic leukemia/ lymphoma Records from patients with T-lymphoblastic leukemia/ lymphoma (T-LL) were also reviewed over the same time period. Twenty-five new diagnoses of T-LL were reviewed; 22 had peripheral blood flow cytometry at CHOA. However, none of these patients presented with a peripheral blood blast count of 1%. Percent monocytes from the flow cytometry report was recorded, similar to the aforementioned groups. Multiparameter flow cytometry Four-color flow cytometric immunophenotyping was performed with a broad panel of antibodies, evaluating for B-lymphoid, T-lymphoid, and myeloid disease. The pertinent antibodies used for diagnosis of B-LL and for monocytes are detailed in Table 1. Peripheral blood samples collected in ethylenediaminetetraacetic acid were adjusted to a cell concentration of between 5000 and 10 000 cells/mL. The peripheral blood sample was then washed 3 times to remove plasma and platelets and resuspended in cell wash (phosphate-buffered saline, fetal bovine serum, sodium azide). Fifty microliters of this sample were added to each tube containing the specific cocktail of antibodies, and the cells were incubated in the dark for 15 minutes. Ammonium chloride was then added to lyse the erythrocytes, and the tubes were incubated in the dark for another 15 minutes. The tubes were then centrifuged, decanted, and washed twice in cell wash, and 500 mL of 1% reagent-grade formalin were added to fix the cells. Acquisition of 10 000–200 000 cells/tube was performed on a BD Canto II flow cytometer and analyzed using Diva software. For each antibody, negative staining levels were set by the use of an isotype-matched control.
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Figure 1. Sample gating of monocytes. A large gate is drawn on forward-scattered light (FSC) vs side-scattered light (SSC) to include all mononuclear cells as well as some granulocytes. Monocytes are then gated from CD64 vs CD33, which separates them from the granulocytes, which show dimmer CD64 and CD33 expression. Percent monocytes are expressed using all events (including all white cells) as the denominator.
Gating strategy To assess percentage monocytes, a gate was placed on forward-scattered light (FSC) vs side-scattered light (SSC) to include all mononuclear cells and a portion of the granulocytes. The monocytes were then gated using a combination of CD64 vs CD33, which allows separation from the normal granulocytes, which express dim CD64 and CD33 (see Fig. 1). The percent monocytes were obtained using all events, to include all white blood cells as the denominator. Complete blood count with differential The complete blood count with differential (CBCD) was performed on a Siemens Advia 120 or 2120 instrument (Malvern, PA). The AMC was calculated by multiplying the white blood cells by the percentage of monocytes and expressed as cells/microliter. In most cases, for both the leukemic and negative control groups, the monocyte percentage was generated by manual differential. Statistical analysis The Wilcoxon test was used to compare percent monocytes and AMCs in the leukemic groups and the negative control group. Significance of the reported P values was defined as P , 0.05.
RESULTS Study group Clinical characteristics of the patients presenting with 1% circulating blasts are detailed in Table 2. This group included 9 girls and 6 boys ranging from 10 months–15 years
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of age. No recurring cytogenetic abnormalities were noted. The clinical characteristics, types of cytopenias, and final diagnoses of the negative control group are detailed in Table 3. There was no statistically significant difference in the ages (P 5 0.663, Wilcoxon test) or sexes (P 5 0.94, chisquare test) between the leukemia group and the negative controls. Flow cytometry The monocytes from the patients with B-LL with 1% blasts averaged 0.8% and were statistically lower than the control group, which averaged 7.1% (Wilcoxon test; P , 0.001) (Fig. 2a). Eleven of the 14 (79%) patients with BLL with 1% blasts had monocyte counts that were below 1%, compared to only 3 patients (8%) in the control group. Follow-up examinations revealed that each of these 3 control patients with monocytes ,1% had a marrow production defect: 2 had aplastic anemia, and 1 had severe vitamin B12 deficiency. Peripheral blood flow cytometry showed monocytes ,1% in 31 of the 52 (60%) of patients with ‘‘typical’’ newly diagnosed B-LL, who had .25% circulating blasts. Fourteen (27%) had monocytes of 1%, and 6 (12%) showed monocytes of 2%. One case (2%) had monocytes .3%. Similarly, among the patients newly diagnosed with T-LL, monocyte percentages were ,1% in 8 of 22 (36%), 51% in 7 (32%), 2% in 3 (14%), and 3% in 4 (18%). Absolute monocyte count The AMC was calculated for each patient in the leukemia and control groups. AMC in the patients with leukemia
Table 2. Clinical characteristics of the B-lymphoblastic leukemia group with 1% circulating blasts by flow cytometry Cytopenias Patient
Age (years)
Sex
Hgb
Plt
ANC
% monocytes
AMC
% blasts
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
9 10 months 15 3 8 10 5 1 2 7 9 5 8 2 12
F F F M M F F M F M M F M F F
7.1 8.6 7.0 2.6 Nl 7.1 10.3 2.6 3.4 8.5 Nl 9.5 9.3 4.9 7.0
90 23 108 23 Nl 87 131 37 32 Nl 60 Nl 93 ,10 Nl
300 1150 240 880 Nl 360 Nl 0 0 490 0 750 250 20 30
0.4 1.3 0.2 0.2 3.5 0.5 0.7 0.5 0.8 0.9 N/A 0.1 1.0 0.7 0.6
31 46 0 0 361 10 0 63 47 87 12 0 33 21 0
0.8 1 0.4 0.5 0.5 0.3 0.7 0.3 0.2 0.8 0.7 0.1 0.7 0.9 0.08
Hemoglobin (Hgb) expressed in g/dL, platelets (Plt) in 3 103/mL, and absolute monocyte count (AMC) and absolute neutrophil count (ANC) in cells/mL. Nl indicates value within the normal range; N/A, not able to be performed.
was statistically lower than AMC in the control group (Wilcoxon test; P , 0.001), Fig. 2b). The AMC was ,100 cells/mL in 14 of 15 patients (93%) of the leukemia group, whereas only 11 of 39 (28%) of the control patients had AMCs at this level.
DISCUSSION Peripheral blood flow cytometry is performed routinely at our institution in cases ranging from unexplained pancytopenia to overt leukemia. While this practice varies among institutions, we have found that establishing the type of leukemia prior to performing the marrow allows for several advantages. These include obtaining proper Children’s Oncology Group consent, drawing appropriate study tubes, ordering appropriate cytogenetics and fluorescence in situ hybridization panels, and placing the correct port for therapy at the time of bone marrow aspiration. This obviates a second bone marrow biopsy or repeat sedation for port placement. It is therefore essential that the pathologist interpreting the flow cytometry be thorough in the evaluation to minimize false-negative results. Monocytes should comprise at least 4% of total white blood cells, with an AMC of at least 180 cells/mL (0.18 3 109 cells/L) in patients older than 2 months [6]. Circulating monocytes provide a window into bone marrow production, as they are often increased first in marrow recovery, with neutrophils following [4]. Monocytes only circulate for 12–24 hours before entering tissue [7], compared to the relatively longer 5.4-day circulation of neutrophils [8], making monocytes a more sensitive indicator of marrow production. Although both neutrophils and monocytes are typically decreased in leukemia, decreased monocytes are more informative for occult leukemia, as many of the nonneoplastic conditions that lead to neutropenia in
children, such as infections, cyclic neutropenia, and autoimmune neutropenia, often cause a relative increase in circulating monocytes [4]. Decreased monocytes are not often seen in children and most often indicate a bone marrow failure syndrome, such as aplastic anemia. Monocytopenia may also be seen in the settings of glucocorticoid administration, sepsis, and hemodialysis [5]. To determine whether these factors were involved in the monocytopenia observed in the leukemic patients, medical records were reviewed at presentation. Patient 6 presented in sepsis and later grew Pseudomonas in her blood culture. This patient died during induction chemotherapy. All the other patients in the occult leukemia group were previously well, with no signs of sepsis and no history of steroids or hemodialysis. It was not only patients with low circulating blasts who had monocytopenia. In fact, the usual presentation of ‘‘typical’’ B-LL, with circulating blasts .25%, also showed a decrease in monocytes. A review of 52 peripheral blood flow cytometry reports revealed that 98% of newly diagnosed B-LL with circulating blasts of .25% showed a monocytopenia of 2%, well below the 4% lower limit of the normal range defined by the American Association of Clinical Chemistry [6]. The reason for the decrease in circulating monocytes in B-LL is unclear. One hypothesis is that the increased blasts in the marrow space are myelophthisic, leading to an overall decrease in hematopoiesis by replacing the bone marrow and excluding the normal marrow elements. However, review of the bone marrow aspirates in the 15 patients in our study with low (1%) circulating blasts revealed that the average blast count was 60%, and half of the cases had easily identifiable background hematopoiesis. In keeping with this finding, the CBCD of these patients occasionally showed mildly decreased counts in only 1–2
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Table 3. Demographics and final diagnoses of negative control group, which had negative peripheral blood flow cytometry Cytopenias Patient
Age (years)
Sex
Hgb
Plt
ANC
% monocytes
AMC
Diagnosis
1 2 3 4 5 6 7
3 1 13 15 9 months 2 6 months
M F M F M F M
10.5 6.0 Nl 5.5 Nl 5.1 Nl
,10 Nl ,10 17 12 11 Nl
Nl 580 40 Nl 910 270 0
4.9 3.3 33.9 7.0 5.9 0.5 13.8
205 82 383 1135 588 0 806
8 9 10 11 12 13 14 15 16 17
14 3 10 months 14 1 1 15 10 7 12
M F F F F F M M F M
7.3 7.9 9.1 4.9 3.8 3.3 Nl 5.9 6.1
Nl ,10 ,10 70 Nl Nl 31 Nl 94 Nl
Nl 160 Nl 1360 1080 Nl 620 Nl 1070 Nl
1.7 0.7 6.3 4.8 1.9 1.9 6.4 4.5 8.4 9.6
32 0 585 167 64 1792 64 1342 86 1563
18 19 20
16 1 11
M M M
4.5 7.7 11.2
118 Nl Nl
Nl 890 1110
0.1 4.2 10.4
0 343 394
21 22 23 24 25 26 27 28 29 30
6 1 16 16 5 13 17 16 15 1
M F M F F M F F F F
9.9 10.1 4.3 11.3 Nl 12.7 Nl 6.3 9.8 8.3
,10 101 93 126 54 ,10 ,10 Nl 33 Nl
Nl 620 1600 450 Nl 370 Nl 320 Nl 450
8.2 3.2 8.1 4.0 12.5 26 10.2 3.1 4.7 24
369 164 151 286 225 346 374 290 0 1202
31 32 33 34 35 36 37
2 9 4 9 14 7 7
F F F M F F M
7.3 10.4 Nl Nl 10.2 10.3 Nl
,10 19 32 11 Nl Nl 75
780 540 Nl Nl 610 Nl Nl
1.7 3.4 7.2 3.3 2.9 3.6 8.9
120 54 955 598 97 120 345
F M
Nl 7.7
15 Nl
Nl Nl
5.1 7.7
879 199
ITP Possible TEC ITP, possible virus TTP ITP SAA Autoimmune neutropenia with antigranulocyte antibodies HIV, disseminated MAI SAA ITP Drug-induced marrow suppression Pearson’s syndrome Autoimmune hemolytic anemia Ehrlichia chaffeensis infection Kawasaki disease Probably viral infection New diagnosis of sickle cell disease with pain crisis Severe vitamin B12 deficiency TEC Benign ethnic neutropenia with viral illness ITP Bacteremia PNH Proprionic acidemia and viral infection Viral illness ITP with possible viral syndrome ITP Sickle cell disease with viral illness ITP and acute appendicitis Pneumonia, multiple infections, immunodeficiency SAA SAA ITP ITP secondary to EBV infection Antigranulocyte antibodies, likely SLE Possible immunodeficiency Autoimmune hepatitis with hypersplenism and sequestration ITP Neutropenia, resolved without therapy
38 39
1 2 months
Hemoglobin (Hgb) expressed in g/dL, platelets (Plt) 3 103/mL, and absolute monocyte count (AMC) and absolute neutrophil count (ANC) in cells/ml. Nl indicates value within the normal range; ITP, idiopathic thrombocytopenic purpura; TEC, transient erythroblastopenia of childhood; TTP, thrombotic thrombocytopenic purpura; SAA, severe aplastic anemia; MAI, Mycobacterium avium intracellularae; PNH, paroxysmal nocturnal hemoglobinuria; EBV, Epstein-Barr virus; and SLE, systemic lupus erythematosus.
lineages. Patient 5 with B-LL even had a normal CBCD, with an absolute neutrophil count of 7000 cells/mL; however, the monocytes were still mildly decreased at 3%. Other possibilities for monocytopenia associated with B-LL include dysregulation of endogenous granulocyte-macrophage colony-stimulating factor or increased endogenous glucocorticoids, both of which
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may lead to decreased monocyte production. The mechanism may be similar to that seen in hairy cell leukemia, which is also poorly understood, but may involve decreased levels of stimulatory cytokines or secretion of inhibitory factors by the malignant hairy cells [9]. Clearly, more study is necessary to elucidate the cause of this relationship.
Figure 2. a. Box plots of the percentage monocytes as determined by peripheral blood flow cytometry for patients with Blymphoblastic leukemia (B-LL) vs the negative control group. The top, middle, and bottom bars of the box represent the 75th, 50th (median), and 25th percentiles, respectively. The lines drawn from the box represent 1.53 intraquartile range (75th percentile–25th percentile). The Wilcoxon test showed that the B-LL group had statistically fewer monocytes than the negative controls (P , 0.0001). b. Box plots comparing the absolute monocyte counts (AMCs) in patients with Blymphoblastic leukemia (B-LL) and in the negative control group. These plots are derived in a similar way as a. The Wilcoxon test showed that the B-LL group had statistically lower AMCs than the negative control group (P , 0.0001).
To assess whether a similar finding is observed with TLL, all cases of T-LL newly diagnosed during the same 3year period were reviewed. Twenty-five new cases were reviewed, of which 22 had peripheral blood flow cytometry at CHOA. However, none of these cases had circulating blasts 1%. By flow report, monocytes were 2% in 82% of newly diagnosed T-LL cases, which suggests a similar biological mechanism to the monocyte suppression seen in B-LL. In more than 85% of newly diagnosed B-LL cases, establishing the diagnosis on peripheral blood flow cytometry is straightforward, with circulating blasts .1%. However, circulating blasts are occasionally ,1%, and sometimes even ,0.1%, making the diagnosis challenging, even with flow cytometry. Decreased monocytes provide a clue to a defect in marrow production and may be useful in diagnosing occult leukemia. The results of our study suggest that if monocytes are ,1% on peripheral blood flow cytometry in the setting of cytopenias or clinical suspicion of leukemia, it is optimal to acquire as many cells as possible in the B-cell tubes to look carefully for rare blasts. Practically, we try to acquire at least 100 000 cells in our lab before calling a result negative. Even if the flow cytometry is still negative, a bone marrow examination should be considered in these cases to evaluate for a marrow production defect. Peripheral blood flow cytometry may be bypassed if the AMC is ,100 cells/ mL with high clinical suspicion of leukemia, and consideration given to proceed with bone marrow examination.
ACKNOWLEDGMENT The authors would like to sincerely thank Traci Leong for her expertise in the statistical analysis of our data. REFERENCES 1. Swerdlow SH, Campo E, Harris NL, et al. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues, 4th ed. Lyon: International Agency for Research on Cancer, 2008. 2. Allan A, Keeney M. Circulating tumor cell analysis: technical and statistical considerations for application to the clinic. J Oncol 2010; 2010:426218. 3. Stetler-Stevenson M, Ahmad E, Barnett D, et al. Clinical Flow Cytometric Analysis of Neoplastic Hematolymphoid Cells; Approved Guideline, 2nd ed. Wayne, PA: Clinical and Laboratory Standards Institute, 2007. 4. Foucar K. Monocytosis. In: Kjeldsberg CR, ed. Practical Diagnosis of Hematologic Disorders, Volume 1, 4th ed. Singapore: American Society for Clinical Pathology, 2006;219–226. 5. Reichard K. Non-neoplastic granulocytic and monocytic disorders, excluding neutropenia. In: Foucar K, Reichard K, Czuchlewski D. Bone Marrow Pathology, 3rd ed. Singapore: American Society for Clinical Pathology, 2010;181–205. 6. Soldin S, Wong EC, Brugnara C, Soldin O. Pediatric Reference Intervals, 7th ed. Washington, DC: American Association of Clinical Chemistry Press, 2011. 7. Glassy E, ed. Color Atlas of Hematology; An Illustrated Field Guide Based on Proficiency Testing. Northfield, IL: College of American Pathologists, 1998. 8. Pillay J, den Braber I, Vrisekoop N, et al. In vivo labeling with 2H2O reveals a human neutrophil lifespan of 5.4 days. Blood 2010;4:625–627. 9. Burthem J, Cawley JC. Hairy Cell Leukaemia. London: SpringerVerlag, 1996.
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Monocytopenia as a Diagnostic Clue to Pediatric B-Lymphoblastic Leukemia with Rare Circulating Blasts SUNITA I. PARK*
AND
BEVERLY B. ROGERS
Children’s Healthcare of Atlanta, 1405 Clifton Road, NE, Egleston Children’s Hospital, Department of Pathology, First Floor, Tower One, Atlanta, GA 30322, USA
Received April 12, 2013; accepted June 27, 2013; published online July 1, 2013.
ABSTRACT
INTRODUCTION
B-lymphoblastic leukemia/lymphoma (B-LL) is the most common childhood cancer. Circulating blasts in the peripheral blood may be rare (1%) and missed, even when flow cytometric immunophenotyping is performed, leading to a false-negative report. The records from all patients with a new diagnosis of B-LL between January 2009 and December 2011 at our institution were reviewed. Of 130 cases with peripheral blood flow cytometry, 15 had a blast count of 1%, with 14 having electronic files for gating monocytes. The percentage of monocytes by flow cytometry and absolute monocyte counts (AMCs) were compared with peripheral blood samples that were negative by flow cytometry, sent due to cytopenia of at least 1 lineage (n 5 39). The monocytes from the patients with leukemia averaged 0.8% and were statistically fewer than the negative controls, which averaged 7.1% (P , 0.001). Eleven of the 14 (79%) patients with leukemia had monocytes ,1%, compared to only 3 (8%) of the negative controls. The AMCs were also significantly lower (P , 0.001), with 93% of the leukemia group having an AMC ,100 cells/mL, compared to only 28% of the negative controls. In patients with cytopenias, percentage of monocytes may be an important diagnostic clue in determining the presence of occult leukemia. If flow cytometry is performed, acquisition of more than the standard 10 000 events is necessary to adequately assess for leukemia. If monocytes are ,1% by flow cytometry in the setting of cytopenias, bone marrow examination is recommended, even with negative peripheral blood flow cytometry.
B-lymphoblastic leukemia/lymphoma (B-LL) is the most common childhood tumor, with an estimated 3000 new cases annually in the United States [1]. The majority of patients present with cytopenias, and blasts are easily seen on peripheral blood smear. Flow cytometric immunophenotyping of the peripheral blood is then performed to characterize the immunophenotype of the blasts, which often express dimmer CD45 than mature B cells and may aberrantly express CD10, CD34, CD58, and terminal deoxynucleotidyl transferase, with lack of surface kappa or lambda staining [1]. Given the expected absence of normal B-cell progenitors (hematogones) in the peripheral blood, which may have a similar phenotype, the leukemic blasts are typically easy to identify by flow cytometry. Rarely, patients present with a very low circulating blast burden, which may be 1%, making the diagnosis challenging, even with peripheral blood flow cytometry. In a paper describing a rare event analysis, Allan and Keeney [2] showed that, given a coefficient of variation of 20% in detecting a positive population, 25 events will define an abnormal cluster of cells. If a sensitivity of 0.01% is desired, 2.5 3 105 cells need to be acquired [2], which is much greater than the standard 10 000 events/ tube [3] that is used in many flow labs. This level of sensitivity may be challenging to achieve in hypocellular samples, samples with low specimen volume, or samples with debris and/or platelets, which can fall into the CD45 dim region and obscure a minor abnormal population. Failing to detect the B lymphoblasts will lead to a falsenegative flow report, which may lead to a false sense of security and/or delay in examining the bone marrow. Monocytes are myeloid-derived white blood cells that aid in antigen presentation. They circulate briefly in the peripheral blood and then migrate to the tissues, where they mature into various cells of the monocyte/histiocyte/ immune accessory cell system. Monocytes are increased in physiologic conditions, such as during the neonatal period and during marrow recovery from agranulocytosis. They are also increased in reactive conditions, such as
Key words: B-lymphoblastic leukemia, flow cytometry, monocytopenia, pediatric
*Corresponding author, e-mail: [email protected]
Table 1.
Monoclonal antibodies used for immunophenotypic characterization
Monoclonal antibody
Clone
Used for
Source
CD19 CD10 CD34 CD20 CD58 TDT Kappa/lambda CD33 CD64
SJ25C1 HI10a 8G12 L27 1C3 (AICD58.6) HT1, HT4, HT8, HT9 TB28-2/1-155-2 P67.6 22
B-LL B-LL B-LL B-LL B-LL B-LL B-LL Monocytes Monocytes
BD BD BD BD BD Pharmingen Beckman Coulter Dako BD Beckman Coulter
B-LL indicates B-lymphoblastic leukemia; BD, Becton Dickinson; TDT, terminal deoxynucleotidyl transferase.
infection, autoimmune diseases, and certain neoplasms [4]. Decreased monocytes are uncommon, and the differential diagnosis includes marrow failure states, such as aplastic anemia, and glucocorticoid administration, hemodialysis, sepsis, and certain hematologic malignancies, such as hairy cell leukemia [5]. This study examines the role of using decreased monocytes, as determined either by flow cytometry or absolute monocyte count (AMC), as a diagnostic clue to aid in the diagnosis of B-LL with very low circulating blasts.
METHODS Study groups With appropriate institutional review board approval, the records from all patients with a new diagnosis of B-LL at Children’s Healthcare of Atlanta (CHOA) during a 3-year period (January 2009–December 2011) were retrospectively reviewed. Of 171 cases, 130 had peripheral blood flow cytometry performed at our institution.
B-LL with 1% blasts Fifteen of the aforementioned 130 patients had a blast count ranging from 0.08%–1.0% by flow cytometry. In all cases, a bone marrow biopsy confirmed the diagnosis of B-LL by both morphology and flow cytometry. The percentage of monocytes, as determined by flow cytometry and AMC, was compared to that of the negative control group. Fourteen of the 15 cases had electronic files that allowed for re-gating of the monocytes by CD33 and CD64 (see following).
Controls This group was made up of all patients seen in 2011 whose peripheral blood was sent for flow cytometry because of cytopenia of at least 1 lineage stated in the clinical history. Of 43 total cases, 39 were included in the study, as they had electronic files that allowed gating of the monocytes. Patients with a prior diagnosis of a hematologic malignancy, solid tumor, or Down syndrome were excluded. Clinical follow-up and bone marrow examination, when performed, were negative for leukemia.
B-LL with .25% blasts Flow cytometry reports from 52 patients with ‘‘typical’’ B-LL who presented with blast counts .25% in the peripheral blood were reviewed. The percent monocytes from the flow cytometry report was recorded, which was generated by gating CD14+ cells with low to intermediate side scatter properties.
Comparison with T-lymphoblastic leukemia/ lymphoma Records from patients with T-lymphoblastic leukemia/ lymphoma (T-LL) were also reviewed over the same time period. Twenty-five new diagnoses of T-LL were reviewed; 22 had peripheral blood flow cytometry at CHOA. However, none of these patients presented with a peripheral blood blast count of 1%. Percent monocytes from the flow cytometry report was recorded, similar to the aforementioned groups. Multiparameter flow cytometry Four-color flow cytometric immunophenotyping was performed with a broad panel of antibodies, evaluating for B-lymphoid, T-lymphoid, and myeloid disease. The pertinent antibodies used for diagnosis of B-LL and for monocytes are detailed in Table 1. Peripheral blood samples collected in ethylenediaminetetraacetic acid were adjusted to a cell concentration of between 5000 and 10 000 cells/mL. The peripheral blood sample was then washed 3 times to remove plasma and platelets and resuspended in cell wash (phosphate-buffered saline, fetal bovine serum, sodium azide). Fifty microliters of this sample were added to each tube containing the specific cocktail of antibodies, and the cells were incubated in the dark for 15 minutes. Ammonium chloride was then added to lyse the erythrocytes, and the tubes were incubated in the dark for another 15 minutes. The tubes were then centrifuged, decanted, and washed twice in cell wash, and 500 mL of 1% reagent-grade formalin were added to fix the cells. Acquisition of 10 000–200 000 cells/tube was performed on a BD Canto II flow cytometer and analyzed using Diva software. For each antibody, negative staining levels were set by the use of an isotype-matched control.
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Figure 1. Sample gating of monocytes. A large gate is drawn on forward-scattered light (FSC) vs side-scattered light (SSC) to include all mononuclear cells as well as some granulocytes. Monocytes are then gated from CD64 vs CD33, which separates them from the granulocytes, which show dimmer CD64 and CD33 expression. Percent monocytes are expressed using all events (including all white cells) as the denominator.
Gating strategy To assess percentage monocytes, a gate was placed on forward-scattered light (FSC) vs side-scattered light (SSC) to include all mononuclear cells and a portion of the granulocytes. The monocytes were then gated using a combination of CD64 vs CD33, which allows separation from the normal granulocytes, which express dim CD64 and CD33 (see Fig. 1). The percent monocytes were obtained using all events, to include all white blood cells as the denominator. Complete blood count with differential The complete blood count with differential (CBCD) was performed on a Siemens Advia 120 or 2120 instrument (Malvern, PA). The AMC was calculated by multiplying the white blood cells by the percentage of monocytes and expressed as cells/microliter. In most cases, for both the leukemic and negative control groups, the monocyte percentage was generated by manual differential. Statistical analysis The Wilcoxon test was used to compare percent monocytes and AMCs in the leukemic groups and the negative control group. Significance of the reported P values was defined as P , 0.05.
RESULTS Study group Clinical characteristics of the patients presenting with 1% circulating blasts are detailed in Table 2. This group included 9 girls and 6 boys ranging from 10 months–15 years
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of age. No recurring cytogenetic abnormalities were noted. The clinical characteristics, types of cytopenias, and final diagnoses of the negative control group are detailed in Table 3. There was no statistically significant difference in the ages (P 5 0.663, Wilcoxon test) or sexes (P 5 0.94, chisquare test) between the leukemia group and the negative controls. Flow cytometry The monocytes from the patients with B-LL with 1% blasts averaged 0.8% and were statistically lower than the control group, which averaged 7.1% (Wilcoxon test; P , 0.001) (Fig. 2a). Eleven of the 14 (79%) patients with BLL with 1% blasts had monocyte counts that were below 1%, compared to only 3 patients (8%) in the control group. Follow-up examinations revealed that each of these 3 control patients with monocytes ,1% had a marrow production defect: 2 had aplastic anemia, and 1 had severe vitamin B12 deficiency. Peripheral blood flow cytometry showed monocytes ,1% in 31 of the 52 (60%) of patients with ‘‘typical’’ newly diagnosed B-LL, who had .25% circulating blasts. Fourteen (27%) had monocytes of 1%, and 6 (12%) showed monocytes of 2%. One case (2%) had monocytes .3%. Similarly, among the patients newly diagnosed with T-LL, monocyte percentages were ,1% in 8 of 22 (36%), 51% in 7 (32%), 2% in 3 (14%), and 3% in 4 (18%). Absolute monocyte count The AMC was calculated for each patient in the leukemia and control groups. AMC in the patients with leukemia
Table 2. Clinical characteristics of the B-lymphoblastic leukemia group with 1% circulating blasts by flow cytometry Cytopenias Patient
Age (years)
Sex
Hgb
Plt
ANC
% monocytes
AMC
% blasts
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
9 10 months 15 3 8 10 5 1 2 7 9 5 8 2 12
F F F M M F F M F M M F M F F
7.1 8.6 7.0 2.6 Nl 7.1 10.3 2.6 3.4 8.5 Nl 9.5 9.3 4.9 7.0
90 23 108 23 Nl 87 131 37 32 Nl 60 Nl 93 ,10 Nl
300 1150 240 880 Nl 360 Nl 0 0 490 0 750 250 20 30
0.4 1.3 0.2 0.2 3.5 0.5 0.7 0.5 0.8 0.9 N/A 0.1 1.0 0.7 0.6
31 46 0 0 361 10 0 63 47 87 12 0 33 21 0
0.8 1 0.4 0.5 0.5 0.3 0.7 0.3 0.2 0.8 0.7 0.1 0.7 0.9 0.08
Hemoglobin (Hgb) expressed in g/dL, platelets (Plt) in 3 103/mL, and absolute monocyte count (AMC) and absolute neutrophil count (ANC) in cells/mL. Nl indicates value within the normal range; N/A, not able to be performed.
was statistically lower than AMC in the control group (Wilcoxon test; P , 0.001), Fig. 2b). The AMC was ,100 cells/mL in 14 of 15 patients (93%) of the leukemia group, whereas only 11 of 39 (28%) of the control patients had AMCs at this level.
DISCUSSION Peripheral blood flow cytometry is performed routinely at our institution in cases ranging from unexplained pancytopenia to overt leukemia. While this practice varies among institutions, we have found that establishing the type of leukemia prior to performing the marrow allows for several advantages. These include obtaining proper Children’s Oncology Group consent, drawing appropriate study tubes, ordering appropriate cytogenetics and fluorescence in situ hybridization panels, and placing the correct port for therapy at the time of bone marrow aspiration. This obviates a second bone marrow biopsy or repeat sedation for port placement. It is therefore essential that the pathologist interpreting the flow cytometry be thorough in the evaluation to minimize false-negative results. Monocytes should comprise at least 4% of total white blood cells, with an AMC of at least 180 cells/mL (0.18 3 109 cells/L) in patients older than 2 months [6]. Circulating monocytes provide a window into bone marrow production, as they are often increased first in marrow recovery, with neutrophils following [4]. Monocytes only circulate for 12–24 hours before entering tissue [7], compared to the relatively longer 5.4-day circulation of neutrophils [8], making monocytes a more sensitive indicator of marrow production. Although both neutrophils and monocytes are typically decreased in leukemia, decreased monocytes are more informative for occult leukemia, as many of the nonneoplastic conditions that lead to neutropenia in
children, such as infections, cyclic neutropenia, and autoimmune neutropenia, often cause a relative increase in circulating monocytes [4]. Decreased monocytes are not often seen in children and most often indicate a bone marrow failure syndrome, such as aplastic anemia. Monocytopenia may also be seen in the settings of glucocorticoid administration, sepsis, and hemodialysis [5]. To determine whether these factors were involved in the monocytopenia observed in the leukemic patients, medical records were reviewed at presentation. Patient 6 presented in sepsis and later grew Pseudomonas in her blood culture. This patient died during induction chemotherapy. All the other patients in the occult leukemia group were previously well, with no signs of sepsis and no history of steroids or hemodialysis. It was not only patients with low circulating blasts who had monocytopenia. In fact, the usual presentation of ‘‘typical’’ B-LL, with circulating blasts .25%, also showed a decrease in monocytes. A review of 52 peripheral blood flow cytometry reports revealed that 98% of newly diagnosed B-LL with circulating blasts of .25% showed a monocytopenia of 2%, well below the 4% lower limit of the normal range defined by the American Association of Clinical Chemistry [6]. The reason for the decrease in circulating monocytes in B-LL is unclear. One hypothesis is that the increased blasts in the marrow space are myelophthisic, leading to an overall decrease in hematopoiesis by replacing the bone marrow and excluding the normal marrow elements. However, review of the bone marrow aspirates in the 15 patients in our study with low (1%) circulating blasts revealed that the average blast count was 60%, and half of the cases had easily identifiable background hematopoiesis. In keeping with this finding, the CBCD of these patients occasionally showed mildly decreased counts in only 1–2
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Table 3. Demographics and final diagnoses of negative control group, which had negative peripheral blood flow cytometry Cytopenias Patient
Age (years)
Sex
Hgb
Plt
ANC
% monocytes
AMC
Diagnosis
1 2 3 4 5 6 7
3 1 13 15 9 months 2 6 months
M F M F M F M
10.5 6.0 Nl 5.5 Nl 5.1 Nl
,10 Nl ,10 17 12 11 Nl
Nl 580 40 Nl 910 270 0
4.9 3.3 33.9 7.0 5.9 0.5 13.8
205 82 383 1135 588 0 806
8 9 10 11 12 13 14 15 16 17
14 3 10 months 14 1 1 15 10 7 12
M F F F F F M M F M
7.3 7.9 9.1 4.9 3.8 3.3 Nl 5.9 6.1
Nl ,10 ,10 70 Nl Nl 31 Nl 94 Nl
Nl 160 Nl 1360 1080 Nl 620 Nl 1070 Nl
1.7 0.7 6.3 4.8 1.9 1.9 6.4 4.5 8.4 9.6
32 0 585 167 64 1792 64 1342 86 1563
18 19 20
16 1 11
M M M
4.5 7.7 11.2
118 Nl Nl
Nl 890 1110
0.1 4.2 10.4
0 343 394
21 22 23 24 25 26 27 28 29 30
6 1 16 16 5 13 17 16 15 1
M F M F F M F F F F
9.9 10.1 4.3 11.3 Nl 12.7 Nl 6.3 9.8 8.3
,10 101 93 126 54 ,10 ,10 Nl 33 Nl
Nl 620 1600 450 Nl 370 Nl 320 Nl 450
8.2 3.2 8.1 4.0 12.5 26 10.2 3.1 4.7 24
369 164 151 286 225 346 374 290 0 1202
31 32 33 34 35 36 37
2 9 4 9 14 7 7
F F F M F F M
7.3 10.4 Nl Nl 10.2 10.3 Nl
,10 19 32 11 Nl Nl 75
780 540 Nl Nl 610 Nl Nl
1.7 3.4 7.2 3.3 2.9 3.6 8.9
120 54 955 598 97 120 345
F M
Nl 7.7
15 Nl
Nl Nl
5.1 7.7
879 199
ITP Possible TEC ITP, possible virus TTP ITP SAA Autoimmune neutropenia with antigranulocyte antibodies HIV, disseminated MAI SAA ITP Drug-induced marrow suppression Pearson’s syndrome Autoimmune hemolytic anemia Ehrlichia chaffeensis infection Kawasaki disease Probably viral infection New diagnosis of sickle cell disease with pain crisis Severe vitamin B12 deficiency TEC Benign ethnic neutropenia with viral illness ITP Bacteremia PNH Proprionic acidemia and viral infection Viral illness ITP with possible viral syndrome ITP Sickle cell disease with viral illness ITP and acute appendicitis Pneumonia, multiple infections, immunodeficiency SAA SAA ITP ITP secondary to EBV infection Antigranulocyte antibodies, likely SLE Possible immunodeficiency Autoimmune hepatitis with hypersplenism and sequestration ITP Neutropenia, resolved without therapy
38 39
1 2 months
Hemoglobin (Hgb) expressed in g/dL, platelets (Plt) 3 103/mL, and absolute monocyte count (AMC) and absolute neutrophil count (ANC) in cells/ml. Nl indicates value within the normal range; ITP, idiopathic thrombocytopenic purpura; TEC, transient erythroblastopenia of childhood; TTP, thrombotic thrombocytopenic purpura; SAA, severe aplastic anemia; MAI, Mycobacterium avium intracellularae; PNH, paroxysmal nocturnal hemoglobinuria; EBV, Epstein-Barr virus; and SLE, systemic lupus erythematosus.
lineages. Patient 5 with B-LL even had a normal CBCD, with an absolute neutrophil count of 7000 cells/mL; however, the monocytes were still mildly decreased at 3%. Other possibilities for monocytopenia associated with B-LL include dysregulation of endogenous granulocyte-macrophage colony-stimulating factor or increased endogenous glucocorticoids, both of which
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may lead to decreased monocyte production. The mechanism may be similar to that seen in hairy cell leukemia, which is also poorly understood, but may involve decreased levels of stimulatory cytokines or secretion of inhibitory factors by the malignant hairy cells [9]. Clearly, more study is necessary to elucidate the cause of this relationship.
Figure 2. a. Box plots of the percentage monocytes as determined by peripheral blood flow cytometry for patients with Blymphoblastic leukemia (B-LL) vs the negative control group. The top, middle, and bottom bars of the box represent the 75th, 50th (median), and 25th percentiles, respectively. The lines drawn from the box represent 1.53 intraquartile range (75th percentile–25th percentile). The Wilcoxon test showed that the B-LL group had statistically fewer monocytes than the negative controls (P , 0.0001). b. Box plots comparing the absolute monocyte counts (AMCs) in patients with Blymphoblastic leukemia (B-LL) and in the negative control group. These plots are derived in a similar way as a. The Wilcoxon test showed that the B-LL group had statistically lower AMCs than the negative control group (P , 0.0001).
To assess whether a similar finding is observed with TLL, all cases of T-LL newly diagnosed during the same 3year period were reviewed. Twenty-five new cases were reviewed, of which 22 had peripheral blood flow cytometry at CHOA. However, none of these cases had circulating blasts 1%. By flow report, monocytes were 2% in 82% of newly diagnosed T-LL cases, which suggests a similar biological mechanism to the monocyte suppression seen in B-LL. In more than 85% of newly diagnosed B-LL cases, establishing the diagnosis on peripheral blood flow cytometry is straightforward, with circulating blasts .1%. However, circulating blasts are occasionally ,1%, and sometimes even ,0.1%, making the diagnosis challenging, even with flow cytometry. Decreased monocytes provide a clue to a defect in marrow production and may be useful in diagnosing occult leukemia. The results of our study suggest that if monocytes are ,1% on peripheral blood flow cytometry in the setting of cytopenias or clinical suspicion of leukemia, it is optimal to acquire as many cells as possible in the B-cell tubes to look carefully for rare blasts. Practically, we try to acquire at least 100 000 cells in our lab before calling a result negative. Even if the flow cytometry is still negative, a bone marrow examination should be considered in these cases to evaluate for a marrow production defect. Peripheral blood flow cytometry may be bypassed if the AMC is ,100 cells/ mL with high clinical suspicion of leukemia, and consideration given to proceed with bone marrow examination.
ACKNOWLEDGMENT The authors would like to sincerely thank Traci Leong for her expertise in the statistical analysis of our data. REFERENCES 1. Swerdlow SH, Campo E, Harris NL, et al. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues, 4th ed. Lyon: International Agency for Research on Cancer, 2008. 2. Allan A, Keeney M. Circulating tumor cell analysis: technical and statistical considerations for application to the clinic. J Oncol 2010; 2010:426218. 3. Stetler-Stevenson M, Ahmad E, Barnett D, et al. Clinical Flow Cytometric Analysis of Neoplastic Hematolymphoid Cells; Approved Guideline, 2nd ed. Wayne, PA: Clinical and Laboratory Standards Institute, 2007. 4. Foucar K. Monocytosis. In: Kjeldsberg CR, ed. Practical Diagnosis of Hematologic Disorders, Volume 1, 4th ed. Singapore: American Society for Clinical Pathology, 2006;219–226. 5. Reichard K. Non-neoplastic granulocytic and monocytic disorders, excluding neutropenia. In: Foucar K, Reichard K, Czuchlewski D. Bone Marrow Pathology, 3rd ed. Singapore: American Society for Clinical Pathology, 2010;181–205. 6. Soldin S, Wong EC, Brugnara C, Soldin O. Pediatric Reference Intervals, 7th ed. Washington, DC: American Association of Clinical Chemistry Press, 2011. 7. Glassy E, ed. Color Atlas of Hematology; An Illustrated Field Guide Based on Proficiency Testing. Northfield, IL: College of American Pathologists, 1998. 8. Pillay J, den Braber I, Vrisekoop N, et al. In vivo labeling with 2H2O reveals a human neutrophil lifespan of 5.4 days. Blood 2010;4:625–627. 9. Burthem J, Cawley JC. Hairy Cell Leukaemia. London: SpringerVerlag, 1996.
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