B-Cell Precursors in Normal Pediatric Bone Marrow

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1 HEMATOPATHOLOGY Original Article B-Cell Precursors in Normal Pediatric Bone Marrow CHARLES W. CALDWELL, M.D., PH.D., EDWARD POJE, M.D., AND MARY A. HELIKSON, M.D. A number of studies have been published pertaining to "normal" lymphocyte subsets in bone marrow. However, these studies are based on normal adult marrow or marrows of children with leukemia in remission or other systemic illness. Data on hematologically normal children are lacking. This study demonstrates that, compared with that of adults, bone marrow of hematologically normal children has an increased percentage of B cells and B-cell precursors. Dual-parameter flow cytometric methods demonstrated subpopulations of B cells at various stages of differentiation; the percentage of cells in these subsets is highest in the very young and decreases with increasing age. Caution must be exercised when searching for early leukemic relapse in pediatric marrows because these normal immature B-cell precursors immunophenotypically resemble leukemic blasts. (Key words: B-cell precursors; B cells; Pediatric bone marrow; Flow cytometry) Am J Clin Pathol 1991;95: Flow cytometric analysis of bone marrow has become commonplace in clinical hematopathology laboratories and has a major role in evaluation of lymphohematopoietic malignancies. 1 " 4 Studies from predominantly adult populations have shown that marrow usually contains very low numbers of early B-cell precursors. 5 "" Older children 12 have shown values similar to those in adult marrow. It is known, however, that children undergo a resurgent marrow hyperplasia after chemotherapy, characterized by increased percentages of B cells and B-cell precursors at various stages of cell differentiation. 12 Hematopoietic cells in fetal liver also are known to be enriched with early B-cell precursors. 13 Most important for hematopathologists is that lymphoid leukemias are composed of cells that are immunophenotypically similar, but not necessarily identical, to normal bone marrow B cells and B-cell precursors. 1,214 " 19 Therefore, caution must be exercised in discriminating the precursors of leukemia from cells of normal marrow. Received June 12, 1990; received revised manuscript and accepted for publication August 24, Supported in part by grants from the Fraternal Order of Eagles of Missouri and the Cancer Research Center, Columbia, Missouri. Address reprint requests to Dr. Caldwell: Department of Pathology, University of Missouri School of Medicine, 1 Hospital Drive, Columbia, Missouri While evaluating data related to B-cell production in resurgent hyperplasia, it became clear that immunophenotypic studies were lacking that focused on B-cell precursors in the bone marrow of hematologically normal children. The current study was performed to determine whether age-related changes in B cells and B-cell precursors occur in normal pediatric marrow compared with that of adults. Significant age-related differences were found, predominantly related to B cells and B-cell precursors. Study MATERIALS AND METHODS Population All specimens were obtained in compliance with regulations of the Institutional Review Board of the University of Missouri School of Medicine. After parents provided informed consent, bone marrow aspirates were obtained from the iliac crests of children (ages ten months From the Departments ofpathology and Surgery. University ofmissouri School of Medicine. Columbia, Missouri. to 17 years) undergoing elective surgical procedures for correction of anatomic abnormalities unrelated to systemic disease, such as correction of inguinal or umbilical hernias. All specimens were obtained immediately after administration of anesthesia. Comparison specimens came from adults diagnosed as having either iron deficiency anemia or "anemia of 816

2 CALDWELL, POJE, AND HELIKSON 817 B-Cell Precursors h Normal Marrow chronic disorders," with no evidence of active infection or inflammation. Preparation of Specimens for Analysis Peripheral blood and bone marrow were collected aseptically by standard techniques. 12 Peripheral blood lymphocytes (PBLs) and bone marrow mononuclear cells (MMCs) were enriched by density gradient centrifugation, adjusted to 1 X 10 I0 /L in RPMI 1640 medium, and analyzed within two hours of receipt. Bone marrow smears were examined by light microscopy after staining with May-Griinwald-Giemsa. Cytocentrifuge preparations were examined similarly after density gradient centrifugation. Monoclonal Antibodies and Immunostaining The specificities and sources of monoclonal antibodies (MoAbs) used in this study are listed in Table 1. Several of these were available as both fluorescein isothiocyanate (FITQ- and phycoerythrin (PE)-labeled reagents. A panel of antimyeloid MoAbs was also used to determine the level of myeloid contamination of the lymphoid light scatter gates (data not shown). Aliquots (50 nl) of cell suspensions (1 X 10' /L) and 50 nl of FITC-labeled MoAbs (at saturating concentrations) were incubated at 4 C for 30 minutes and the cells analyzed by flow cytometry Monoclonal FITC-labeled isotypic negative controls were analyzed in parallel with each specimen. CD* CD1 CD2 CD3 CD4 CD5 CD8 CD10 CD19 CD20 CD2I CD45 NC TABLE 1. MONOCLONAL ANTIBODIES USED IN THIS STUDY WITH THEIR CD DESIGNATIONS AND MAIN REACTIVITY RELATED TO THIS PROJECT MoAbf CCT6 CCT11 CCT3 CCT4 Leu-1 CCT8 CALLA Leu-12 Leu-16 B2 HLe-l HLA-DR Specificity Thymocytes Mature T cells, NK cells Mature T cells Helper/inducer T cells Pan T cells, B cell subset Suppressor/cytotoxic T cells Common acute lymphoblastic leukemia antigen Pan B cells Pan B cells Mature B cells, Epstein-Barr virus receptor Common leukocyte, T200 antigen Class II histocompatibility antigen CD = clusters of differentiation, as defined by the Fourth International Workshop on Human Leukocyte Differentiation Antigens 20 : NC = not clustered. t Sources of MoAbs: The CCT series and CD21 (B2) were from Coulter Immunology. Hialeah, Florida: the remaining MoAbs were from Becton Dickinson, Mountain View, California. For dual-parameter staining, 50 /*L of cell suspension was added to 50 nl of FITC-labeled MoAb and 50 nl of PE (or RDl) -labeled MoAb. After incubation at 4 C for 30 minutes, the cell suspension was washed twice with cold phosphate-buffered saline (PBS)/0.01% (weight/volume [w/v]) sodium azide, resuspended in 0.5 ml of cold buffer, and submitted to flow cytometric examination. Combined DNA and MoAb Staining An aliquot of 50 /ih of cells was first immunostained with FITC-labeled CD45, as above, and the cells washed thrice in PBS and resuspended in 1.0 ml of cold 70% (volume/volume [v/v]) ethanol. After fixation for 30 minutes, the cells again were washed thrice with PBS and resuspended in 1.0 ml of PBS containing 50 ng propidium iodide (Sigma Chemical Company, St. Louis, MO). Cells then were submitted to flow cytometric examination after an incubation of one hour in the dark at ambient temperature. Flow Cytometry All examinations were performed with a FACScan (Becton Dickinson, Mountain View, CA) or PROFILE (Coulter Electronics, Hialeah, FL). Technical aspects on standardization of fluorescence-intensity measurements and analysis have been reported For dual-parameter analysis of DNA and CD45, histograms were collected of FITC fluorescence versus propidium iodide fluorescence from 10,000 cells within the light scatter gates. The percentages of cells within the S- and G2 + M phases of the cell cycle were calculated by simply excluding the cells within the G 0 /G peak from each peak of CD45 fluorescence intensity (FI). This was accomplished with a computer-assisted quadrant analysis program (Coulter). RESULTS Peripheral Blood and Bone Marrow Morphologic Characteristics Morphologic review of May-Griinwald-Giemsa-stained smears of peripheral blood and bone marrow revealed an increase in the percentage of "lymphocytes" in children, compared with adults (Table 2). Many of the marrow "lymphoid" cells (small arrows, left panel of Fig. 1) were smaller than peripheral blood lymphocytes and had scant agranular cytoplasm with hyperchromatic, compact nuclei similar, if not identical, to "hematogones." 22 " 24 These Vol. 95 No. 6

3 818 HEMATOPATHOLOGY Original Article TABLE 2. TOTAL NUMBER OF LYMPHOCYTES AND PERCENTAGES OF PERIPHERAL BLOOD LYMPHOID SUBSETS Subjects Total* Lymphs HLA-DR CD 19 CD10 CD20 CD21 CD2 CD4 CD8 Children < 1-5 years (n= 15) 6-10 years (n= 18) years (n= 12) Adults (n = 46) 5.6 (1.9)* 3.3 (1.2) 2.8 (0.9) 2.9 (0.6) 22.2f (6.8) 15.1 (3.8) 11.8 (3.2) 8.9 (3.9) (4.2) 8.1 (1.9) 6.4 (2.9) 0.2 (0.1) 0.4 (0.1) 0.3 (0.2) 0.3 (0.3) 21.0 (4.6) 12.2 (3.4) (2.4) 14.0 (4.6) 10.3 (3.1) 10.4 (2.9) 5.5 (2.2) 66.5 (11.6) 71.3 (10.7) 72.0 (15.3) 78.3 (12.7) 38.3 (6.2) 39.1 (5.8) 41.5 (7.4) 43.4 (6.3) (5.5) 27.1 (5.7) * The absolute lymphocyte count X I0 9 /I- (mean and standard deviation [in parentheses]), t The mean percentage of cells within the "lymphoid" light scatter gate positive for each MoAb. t The standard deviation for each measurement. small hyperchromatic cells as in earlier studies 22 were not identified in peripheral blood smears from these children. Adult bone marrows contained very few of these cells, but instead consisted of those with typical mature lymphocytic morphologic characteristics. The cell indicated by the larger arrow (left panel of Fig. 1) represents the usual concept of a lymphoblast, with immature nuclear chromatin and small nucleoli. The panel on the right FIG. 1. A. May-Griinwald-Giemsa-stained smear of bone marrow aspirate from a child, illustrating an increase of small, hyperchromatic lymphoid cells with scant agranular cytoplasm and virtual absence of nucleoli (small arrows) and a typical lymphoblast (large arrow). B. A focus of these small hyperchromatic cells commonly encountered in pediatric, but not adult, marrows (X 1,000). A.J.C.P.-June 1991

4 CALDWELL, POJE, AND HELIKSON 819 B-Cell Precursors in Normal Marrow illustrates a focus of small hyperchromatic cells commonly found in the marrows of children but not adults. MoAb-Defined Peripheral Blood Subsets The absolute numbers of total lymphocytes and percentages of B-cell and T-cell subsets are listed in Table 2. As shown, there is a significant difference between values from adults and those from children, as well as an agerelated change, consistent with previous literature. 25 This is most obvious with the CD 19, CD20, and CD74 B-cell subsets. Virtually no immunologically defined immature B cells (CD19 + /CD10 + ) were apparent in the blood of children or adults. It is known that neonates have circulating immature T cells, 26 but because neonates were excluded from the current study, this finding could not be corroborated. MoAb-Defined Bone Marrow Cellular Subsets The percentage of cells with "lymphoid" morphologic characteristics, including "hematogones" and percentages of MoAb positivity of bone marrow cells within the "lymphoid" area of light scatter, are shown in Table 3. Compared with results in adults, the most striking finding is a decreased ratio of T cells to B cells and an increase in immature B cells. By single MoAb analysis, the pediatric marrow cells reactive with B-cell-restricted or B-cell-associated MoAbs are increased in percentage compared with those in adults. CD 19 was present on an average of 44.3% of the lymphoid cells in the youngest children and decreased to 33.4% in the 6-10-year-old group and 22.8% in the year-old children, whereas only 14.5% of the lymphoid cells were CD19 + in adults. A similar pattern was noted with CD 10 and CD20, although the differences were not as great as with CD 19. The overall pattern of immunologic staining in children suggests a "left shift" toward immature B cells (CD 19 +, CD20"), similar to that in fetal bone marrow 13 and that during resurgent hyperplasia. 12 The percentages of CD19 + and CD20 + cells were similar in adult marrows, suggesting mainly mature B cells. Within the T-cell population, there were very few (<0.3%) CD1 + cells or dual CD4 + /CD8 + cells (<0.4%) indicative of immaturity in either adult or pediatric marrow. In adults, the percentage of cells bearing T-cell antigens was 65.6 ± 6.4%, whereas in children the percentage ranged from 33.4 to 59.8%, inversely related to age group. Because virtually no immature T cells (CD1 + ) were present by single-parameter analysis, additional dual-marker studies were limited to B-cell subsets. Dual-parameter staining of B-cell precursors and B cells with combinations of CD45/CD19, CD10/CD19, and CD20/CD19 MoAbs (Fig. 2) revealed that most of the B cells in children were at immature stages of differentiation, based on known immunostaining patterns similar to those observed in resurgent hyperplasia. 12 Our earlier work demonstrated that the brightest CD45 + B cells (peak C, Fig. 2, left panel) were mainly mature slg + cells, that the intermediate CD45 + population (peak B, Fig. 2, left panel) was enriched for cytoplasmic mu-positive pre-b-cells, and that the dimmest CD45 + population (peak A, Fig. 2, left panel) was negative for slg and cytoplasmic mu, but brightly positive for CD 10 (common acute lymphoblastic leukemia antigen), thus corresponding to pre-pre-b-cells. Thus, these three populations appear to represent increasing stages of maturity in B-cell differentiation. Table 4 contains the percentages of B cells in each of these FI peaks. The middle peak (mainly cytoplasmic mu-positive TABLE 3. PERCENTAGES OF CELLS WITH A "LYMPHOID" APPEARANCE AND MoAb-DEFINED BONE MARROW CELLULAR SUBSETS IN CHILDREN OF DIFFERENT AGE GROUPS AND ADULTS Subjects % Total' Lymphs HLA-DR CD19 CD 10 CD20 CD21 CD2 CD4 CD8 Children <l-5 years (n= 15) 6-10 years (n= 18) years (n= 12) Adults (n = 46) 39.6 (16.1)$ 36.4 (12.8) 18.1 (6.2) lt (18.2) 47.4 (11.8) 22.8 (6.2) 17.3 (5.1) 44.3 (12.2) 33.4 (5.6) 22.4 (4.7) 14.5 (3.9) 27.0 (6.9) 17.3 (5.3) 14.6 (4.1) 5.4 (2.2) 29.4 (5.5) 20.7 (5.1) (3.9) 6.3 (4.0) (3.1) 5.8 (2.3) 33.4 (8.8) 51.5 (9.1) 59.8 (8.5) 65.6 (9.4) 19.2 (6.9) 27.3 (6.2) (7.3) 13.1 (4.4) 23.4 (4.9) 20.3 (4.7) 22.3 (5.1) * The percentage {mean and standard deviation [in parentheses]) of total bone marrow mononuclear cells that had a "lymphoid" appearance, including the "hematogones." t The mean percentage of cells within the "lymphoid" light scatter gate positive for each MoAb. $ The standard deviation for each measurement. Vol. 95 No. 6

5 820 HEMATOPATHOLOGY Original Article A Q U 1 CD 45 CD 10 CD 20 FIG. 2. Dual-parameter flow cytometric histograms illustrating subpopulations of B cells and B cell precursors commonly encountered in pediatric marrows. In each panel, the MoAb indicated on the x-axis was FITC labeled, while that on the y-axis was PE labeled. All histograms represent the accumulation of 20,000 cells from within the "lymphoid" light scatter gate. pre-b-cells) is the most prominent in all children (mean, % of lymphoid cells or approximately half of CD19 + cells) and is related inversely to age. Percentages of pre-pre-b-cells and slg+ B cells are similar (means of % and %, respectively) between age groups of children, but are still higher than in adult specimens. CD10 + /CD19 + cells were broken down into CD10 brighl (peak B, Fig. 2, middle panel) and CD10 dim cells (peak A, Fig. 2, middle panel). The CD10 brisht population is thought to consist of the least mature CD10 + cells Again, percentages of these cells are highest in the younger children and decline with age (Table 5). Dual immunostaining with CD20 (a marker of more mature B cells) and CD 19 (Fig. 2, right panel) resulted in histograms with increased percentages of CD19 + but CD2CT cells (peak A, Fig. 2, right panel), thus supporting the immaturity of these cells. In contrast, nearly all B cells in adult marrow are of a mature phenotype (CD19 +, CD20 +, CD21 +, slg+), consisting almost entirely of CD45 bright cells, with only 5.4% CD10 + cells. Only 2 of 21 adult marrows demonstrated evidence of B-cell subpopulations, and these accounted for less than 5% of the total of CD19 + cells. Proliferative Activity of B-cell Subsets As shown in Figure 3, of the three subpopulations of B cells and B-cell precursors defined by different levels of CD45 FI, less than 0.5% of S- and G 2 + M-phase cells were identified in the CD45 bnghi peak, which also contained T cells. The presumption of B-cell-associated DNA content was made, based on the CD45/CD19 dual-parameter histogram pattern shown in the left panel of Figure 2. Peaks A and B, the CD45 dim and CD45 in,crmcdia,c peaks, demonstrate that virtually all cells within these areas of CD45 FI are CD 19 positive, thus strongly suggesting B-cell lineage. The CD45 d,m peak had less than 1% of S- and G 2 + M-phase cells, with the greatest degree TABLE 4. PERCENTAGE OF BONE MARROW B CELLS IN VARIOUS STAGES OF DIFFERENTIATION, AS DEFINED BY CD19/CD45 FI MEASUREMENTS TABLE 5. PERCENTAGES OF CD10 D,m AND CD10 Brlgh * CELLS IN THE BONE MARROWS OF CHILDREN AT DIFFERENT AGES AND OF ADULTS Subjects CD19 CD45""" CD45"" CD45"" i "" Subjects CDIO CD10 0 "" CDIO"" 31 " Child <l-5 years (n= 15) 6-10 years (n= 18) years (n = 12) Adult (n = 46) 44.3 (7.9) 33.4 (7.6) 22.4 (6.9) (2.3) 5.2 (2.6) 4.0 (1.9) 0.2 (0.2) 22.2 (8.8) 14.6 (5.1) 12.1 (5.0) 3.9 (1.9) 6.7 (4.2) 6.9 (3.7) 5.2 (3.2) 5.5 Children <l-5 years (n = 15) 6-10 years (n = 18) years (n = 12) Adults (n = 46) 27.0 (6.9) 17.3 (5.3) 14.6 (4.1) 5.4 (2.2) (6.1) 11.0 (4.8) 4.8 (2.6) 5.4 (3.2) 3.6 (1.6) 3.1 (1.7) 0.3 (0.1) Mean and standard deviation (in parentheses) of percentage MoAb positivity from within the "lymphoid" light scatter gates. CD45 dim, intermediate (Int), and bright refer to the percentage of cells within each of the three peaks of CD45 FI as shown in Figure 2. Mean and standard deviation (in parentheses) of percentage MoAb positivity from within the "lymphoid" light scatter gates. CD10 dim and bright refer to the percentage of cells within each of the two peaks of CDIO FI as shown in Figure 2. A.J.C.P.-June 1991

6 CALDWELL, POJE, AND HELIKSON 821 B-Cell Precursors in Normal Marrow a.255 B i? a E3 IBB 2 2? 12 1? 22 2? 32 3? 42 CONTENT DNA s in a ~ 4 $$ '' < ^ # * - 10" 10' 10' 1B J IB" CD45 EXPRESSION FIG. 3. Dual-parameter flow cytometric histogram of CD45 expression (x-axis) and DNA content (y-axis). Propidium iodide was used to determine DNA cell cycle, while CD45 was FITC labeled. of proliferation, as defined by cell cycle analysis, found in the CD45 imcrmcdia,e peak, which contained 15.1% S- and G 2 + M-phase cells. It was this peak that also contained an enrichment of cytoplasmic-mu-positive pre-b-cells in a previous study. 12 Hematogones in bone marrow sections have been described as nonmitotic, nonnucleolated cells. 22 This is supported by our measurement of the proliferative fraction by cell cycle analysis. DISCUSSION The principal finding of this study is that a normally increased proliferation of B-cell precursors at various stages of differentiation may be found in the marrows of healthy children. Extending the knowledge that percentages of "hematogones" 22 " 24 and B-cell precursors are increased in the young, this study defines age-related normal data as a basis for interpreting these marrow cell subsets. B cells are known to derive in large part from the bone marrow. 27,28 Therefore, it is not surprising that increased percentages of these cells have been found in the bone marrows of children and adults with various diseases that are associated with resurgent hyperplasia Previous work from this laboratory 12 has shown that these cells consist of multiple subpopulations of B-cell precursors at sequential stages of cell differentiation, similar to those seen during in vitro culture of fetal bone marrow. 13 Consistent with our observations supporting age-dependent proliferative activity, young mice have a greater number i of CD45RA + (B220 + ) cells than adult mice. 31 These cells also are predominantly immature B cells. It is unclear why B-cell precursors are present in normal, healthy children in increased percentages compared with adults. This finding may relate to chronic or repetitive exposure to new antigens during childhood, a challenge that declines with age. 27 ' 28 Age-related B-cell proliferation also may be influenced by stromal cells. Studies in mice have shown that marrow stromal cells from young animals better support continued growth and differentiation of B cells than those of older mice. 31 Stromal cells respond to stimulation by various cytokines from activated cells and may participate in the regulation of B-cell expansion that accompanies immune reactions. In a previous study, 12 normal pediatric marrow from the overall control population was found to be similar to that of adults in terms of B-cell precursors. However, three often of these children had small populations of immature B-cell precursors. These patients were four to five years old. All children older than ten years had levels of B cells and B-cell precursors consistent with adult values. This concurs with our observation that B-cell production declines with age, as suggested in Table 3. In general, these findings agree with studies based on postchemotherapy specimens. From the studies of combined CD45 expression and DNA cell cycle, it appears that the most active cells in DNA synthesis are not the "hematogones," but the intermediate CD45 + cells that previously were found to be enriched for cytoplasmic mu heavy chains. 12 This finding is consistent with an earlier report 9 demonstrating most of the proliferating cells of bone marrow in the CD10 + population. In this, and a previous study, 26 we found that this CD45' nlcrmcd ' a,e peak was associated predominantly with the CD10 dim population, whereas the CD45 dim peak was associated with the CD10 bri8h " peak. The CD10 bri8ht peak is probably the main component of "hematogones." 22 It is common to find three peaks of positivity with CD45/CD19 In children. Leukemic cells usually increase the percentage of immature cells in one or more of these peaks. Children are known to have increased "lymphocytes" and morphologically distinct lymphoid cells referred to as "hematogones" 22 " 24 on bone marrow differentials, as well as an increased percentage of circulating B cells. 25 Such "lymphocytes" in the marrow primarily are early B-cell precursors, as demonstrated by cell-sorting experiments (data not shown) and previous work. 12 Flow cytometric histograms have shown the relationship between forward angle light scatter (FALS), a correlate of cell size, and subpopulations of immature B cells. 26 The least mature B-cell subpopulations identified in these normal chil- Vol. 95 No. 6

7 822 HEMATOPATHOLOGY Original Article dren were also the smallest, consistent with the morphologic impression of small, compact "lymphocytes" or marrow "hematogones." 22 " 24 As pointed out by Longacre and associates, 22 a number of investigators have described the small, lymphoid-appearing cells seen in the bone marrow after chemotherapy 32 " 37 and in various diseases 29,30 probably first termed "hematogones" by Vogel and associates It has been proposed that increased numbers of these cells are present in any actively regenerating marrow. 38,39 The current study shows that the number of these immature B-cell precursors in the marrow of healthy children is greater than in adults and declines with age, and that this is an apparently normal phenomenon. The knowledge that healthy children have increased percentages of immature B-cell precursors and lower percentages of T cells than adults implies that using adult marrow as controls for studies in children may be misleading, especially when evaluating pediatric marrows for evidence of leukemic relapse. In this instance, increased levels of immunophenotypically immature B cells bearing the phenotype of malignant blasts may be found 14 ' 5,18 and, depending on the patient's age, may not represent residual or relapsed malignancy. These are important points to consider when evaluating pediatric marrows. Acknowledgments. The authors thank Drs. William Patterson and Yohannes Yesus for helpful suggestions and review of the manuscript. They also thank Susan Souchek, MT(ASCP), Nancy Case, MT(ASCP), Jim Ford, MT(ASCP). Ann Wilson, MT(ASCP), and Pam Puig, MT(ASCP), all of the Flow Cytometry Laboratory, Department of Pathology. REFERENCE 1. 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