Leukocyte morphology in blood and bone marrow *

Background

Leukocytes comprise a wide variety of cell types, and evaluation of these components is critical in medical and clinical laboratory practice. Although sensitive laboratory methods may offer strong clues about leukocyte abnormalities, accurate morphologic evaluation of these cells is key in confirming suspicion for a pathologic process and in guiding clinical decision making. However, proper assessment of these cells requires understanding of normal quantitative and morphologic parameters and the deviations from these that are seen in neoplastic and non-neoplastic states.

Content

This chapter discusses essential aspects of evaluating of leukocyte components most commonly found in peripheral blood and bone marrow specimens. The clinical value of leukocyte morphologic examination will be discussed with an overview of specimen preparation and normal quantitative ranges. Morphologic features of granulocytes, maturing myelomonocytic cells, lymphocytes, plasma cells, and other special cell types will be detailed. Importantly, this chapter describes cytomorphologic changes of such cells representing a spectrum of neoplastic and non-neoplastic conditions.

Peripheral blood

A normal PB film should contain anucleated red blood cells (RBCs), platelets, and leukocytes. The total WBC (or leukocyte) count is composed of mostly neutrophils and lymphocytes with smaller subsets of monocytes, eosinophils, and basophils. This is reflected in the CBC with five-part WBC differential resulted by modern automated hematology analyzers, which also flag the presence of immature granulocytes, blasts, and abnormal lymphocytes. Each leukocyte component is reported as a percentage of total WBCs and also as an absolute number per microliter (μL). Reference WBC intervals may vary depending on the laboratory; sources of variation include characteristics of different patient populations, age, sex, and methodology of testing.

Reference intervals have been established by several studies for their respective populations. ^, WBC reference intervals are largely determined by age and are typically highest at birth (15 to 30 × 10 ⁹ /L), declining eventually at 1 year of age (6.0 to 17.5 × 10 ⁹ /L) with steady levels throughout childhood (4.5 to 15.5 × 10 ⁹ /L, ages 3 to 10; 4.5 to 13.5 × 10 ⁹ /L, ages 10 to 17), while for adult ages, a range of 4.1 to 10.2 × 10 ⁹ /L is commonly accepted. Absolute neutrophil counts (ANCs) are highest at birth, stabilizing into a range of 1.5 or 1.8 to 8.0 × 10 ⁹ /L by childhood and adulthood. Ranges specific for certain ethnicities should be considered because variations can occur in African Americans, with slightly lower reference intervals for both the WBC and the ANC. Parameters for this and other populations with benign variations should be interpreted in the appropriate context to avoid misidentification of leukopenia or neutropenia. A physiologic, but mild, generalized leukocytosis can occur in association with cigarette smoking, increased body mass index, hypertension, hyperlipidemia, diabetes, and decreased alcohol consumption. For additional information on how to establish reference intervals and on reference intervals for hematologic parameters, refer to Chapter 9 and the Appendix, respectively.

Absolute lymphocyte counts (ALCs) fluctuate within the first year of life but stabilize as the predominant leukocyte form in the PB until approximately age 7, when neutrophils again become more prominent. ^, The ALC reference interval in adulthood is approximately 1.0 to 4.0 × 10 ⁹ /L. The adult monocyte range is from 0 to 0.8 × 10 ⁹ /L, eosinophils from 0 to 0.45 × 10 ⁹ /L, and basophils from 0 to 0.2 × 10 ⁹ /L.

Bone marrow

The cellular composition of BM sampling is determined visually and requires inspection of terminally differentiated leukocytes and also their precursor stages. Accepted adult BM intervals for leukocyte components are listed in Table 75.1 . The lymphoid compartment can show significant proportional variation by age, with more abundant lymphocytes in the neonatal period and childhood due to increased precursor cells, ^, and myeloblasts can be minimally higher in pediatric specimens as well.

Differential counts should be performed on BM preparations because this set of data yields information on potential deviations from the typical normal range for a particular lineage, the degree of myeloid maturation, and the myeloid:erythroid (M:E) ratio. Although many laboratories note percentages of individual granulocytic components (i.e., promyelocytes, myelocytes, metamyelocytes, etc.), pathologists in some laboratories opt to combine the entire neutrophilic series into one composite parameter (“myeloid cells”), noting whether there is appropriate progressive maturation, left-shift, or perhaps maturation arrest at a particular stage. The M:E ratio should be calculated by adding granulocytes, monocytes, and their precursors and dividing this sum by total nucleated erythroid precursors. The normal M:E ratio may range from 1.5:1 up to 4:1. Other marrow components that are evaluated but not enumerated include megakaryocytes, mast cells (MCs) (except in MC disease), histiocytes, and, if present on preparations, stromal elements (i.e., adipocytes, osteoblasts, osteoclasts, macrophages, fibroblasts, endothelial cells), and other nonhematopoietic cells.

Normal morphology and stages of maturation

Neutrophilic myelopoiesis (or granulopoiesis) involves maturation of earliest forms (myeloblasts) giving rise to maturing forms with nuclear and cytoplasmic changes, with terminal differentiation to segmented neutrophils. The stages of myelopoiesis include the myeloblast, promyelocyte, myelocyte, metamyelocyte, band, and segmented neutrophil ( Fig. 75.1 ). In normal states, only mature neutrophils (segmented and occasional band forms) are present in the PB, whereas all stages are present in the BM. The following are descriptions of these cells in normal resting marrow states.

The myeloblast (15 to 20 μm) is the most immature recognizable cell in this series ( Fig. 75.1 A). It is medium in size and shows a round to ovoid nucleus with a high nuclear/cytoplasmic (N/C) ratio, finely dispersed chromatin, and one or more conspicuous nucleoli. A thin rim of lightly basophilic cytoplasm is present. Myeloblast forms are typically agranular, although scant azurophilic granules may be present. Auer rods, to be described later, are not a feature of normal myeloblasts.

The promyelocyte (12 to 24 μm) is the largest cell in the maturation sequence and is characterized by basophilic cytoplasm showing a paranuclear hof with few to many primary azurophilic granules ( Fig. 75.1 B). The N/C ratio is not as high as in the myeloblast; nuclei are ovoid and eccentric with chromatin slightly more condensed. Nucleoli are not as prominent in this stage, although they still may be visible. The neutrophilic myelocyte (10 to 18 μm) stage is distinguished by the appearance of secondary (or neutrophilic lineage-specific) granules imparting a mixed pink-basophilic (amphophilic) granular appearance to the cytoplasm, which is now more abundant ( Fig. 75.1 C). Nuclei are still round to ovoid, although very slight indentations can be present. Chromatin is progressively condensed, and a nucleolus is not visible. This is the last maturation stage where precursors can undergo mitosis. The neutrophilic metamyelocyte (10 to 18 μm) exhibits abundant secondary pink granules with fewer azurophilic granules ( Fig. 75.1 D). Characteristically, the nucleus becomes kidney shaped with nuclear indentation, which should be less than one-half of the presumed width if the nucleus were round. Although discrete stages are described, progressive maturation occurs in a continuum, and sometimes cells will be encountered with overlapping features of consecutive stages (i.e., “late” promyelocyte versus “early” myelocyte).

Nuclear lobation and chromatin pattern are significantly more mature at the band neutrophil (or simply, “band”) stage (10 to 18 μm), where the nuclear indentation is more pronounced at over one-half of the nuclear width. The nucleus may take on a horseshoe or other elongated U-, S-, or C-shaped configuration ( Fig. 75.1 E). Bands can be present at up to 10% of peripheral leukocytes and can be increased in inflammatory, infectious, or other physiologic stress states. The cytoplasm is pale with a predominance of specific secondary tan-pink granules similar in hue to background RBCs. Many of the granules may be minute and inconspicuous, but their presence imparts an acidophilic, vaguely granular appearance to the cytoplasm.

The most mature myeloid constituent is the segmented neutrophil (10 to 15 μm), often more simply referred to as a neutrophil, which is the predominant leukocyte form in blood in adults. Although the cytoplasm is similar to that of the band, the nucleus takes on a segmented configuration with division into two to five lobes, which are connected by thin solid thread-like filaments not seen in bands ( Fig. 75.1 F). The majority of neutrophils will have two to four nuclear segments, while five segments are normally only present in less than 5% of circulating forms. A minor subset of neutrophils may have agranular peripheral cytoplasmic clearing with slight surface protrusion, which may signify active motility. Neutrophils in PB films of females can occasionally show small “drumstick”-like nuclear formations consistent with Barr bodies, which are extranuclear chromatin extensions originating from an inactivated X chromosome.

Given the wide spectrum of appearance of bands and neutrophils, not surprisingly, there is morphologic overlap and interpretative subjectivity in distinguishing these forms. Band nuclei can take on quite twisted and tortuous configurations, mimicking neutrophil morphology, but nuclear constriction between segments to a thin filament without visible internal chromatin can help to identify a neutrophil. Although bandemia has been suggested in some studies to be helpful in detecting serious infections in young infants, multiple studies have found no significant clinical utility of separating bands and segmented neutrophils, particularly for patients older than 3 months of age. Therefore the common recommendation, as put forth by the Clinical and Laboratory Standards Institute, is for these forms to be counted together as simply “neutrophils” in PB and BM evaluations.