Case Study

After studying this chapter, the reader should be able to respond to the following case study:

A 35-year-old woman noticed multiple pinpoint red spots and bruises on her arms and legs. The hematologist confirmed the presence of petechiae, purpura, and ecchymoses on her extremities and ordered a complete blood count, prothrombin time, and partial thromboplastin time. The platelet count was 35 × 10⁹/L, the mean platelet volume was 13.2 fL, and the platelets appeared large on the Wright-stained peripheral blood film. Other complete blood count parameters and the coagulation parameters were within normal limits. A Wright-stained bone marrow aspirate revealed 10 to 12 small unlobulated megakaryocytes per low-power field.

Megakaryocytopoiesis

Platelets are anucleate blood cells that circulate in amounts of 150 to 400 × 10⁹/L, with mean counts slightly higher in women than in men.¹ Platelets trigger primary hemostasis on exposure to endothelial, subendothelial, and plasma procoagulants in blood vessel injury. On a Wright-stained wedge-preparation blood film, platelets are distributed throughout the red blood cell monolayer at 7 to 21 per 100× field. They have an average diameter of 2.5 µm, corresponding to a mean platelet volume (MPV) of 8 to 10 fL in an isotonic suspension, as determined using laboratory profiling instruments.² Their internal structure, although complex, is granular but scarcely visible using light microscopy.

Platelets arise from unique bone marrow cells called megakaryocytes. Megakaryocytes are among the largest cells in the body and are polyploid, which means that they possess multiple chromosome copies within a single cell. On a Wright-stained bone marrow aspirate film, each megakaryocyte is 30 to 50 µm in diameter with a multilobulated nucleus and abundant granular cytoplasm. In healthy intact bone marrow tissue, megakaryocytes cluster in the extravascular compartment adjacent to the abluminal membrane (the surface opposite the lumen) of venous sinusoid endothelial cells (Figure 13-1).³ Myelocytic and erythrocytic precursor cells, which locate further from the endothelial cells, may cross the megakaryocyte cytoplasm to reach the sinusoid lumen, a faux phagocytosis known as emperopolesis.⁴ Megakaryocytes are also harvested from the lungs.⁵ In a normal Wright-stained bone marrow aspirate film, the microscopist may identify two to four megakaryocytes per 10× low-power field.

Megakaryocyte Progenitors

The megakaryocyte-erythrocyte progenitor (see Figure 7-13) differentiates into the megakaryocyte lineage under the influence of the hormone thrombopoietin (TPO) and a series of cytokines (see Table 13-3). There are three megakaryocyte lineage-committed progenitor stages, defined by their culture colony characteristics. In order of differentiation, these are the least mature burst-forming unit (BFU-Meg), the colony-forming unit (CFU-Meg), and the most mature light-density CFU (LD-CFU-Meg).⁷ All three progenitor stages resemble small lymphocytes and cannot be distinguished by Wright-stained light microscopy. The BFU-Meg and CFU-Meg are diploid and participate in normal mitosis, maintaining a pool of megakaryocyte progenitors. Their proliferative (mitotic) properties are reflected in their ability to form colonies of hundreds (BFUs) or scores (CFUs) of progeny in culture (Figure 13-2). In contrast to the BFU-Meg and CFU-Meg, the LD-CFU-Meg has little proliferative capacity and produces few cells, but begins the progress through endomitosis to reach increased nuclear ploidy.

The LD-CFU-Meg may be a transitional, or “promegakaryoblast,” stage in which polyploidy is first established, but its morphology is indistinguishable from that of small lymphocytes. At the LD-CFU-Meg point of development, megakaryocyte progenitors enter terminal differentiation, as they lose their ability to undergo normal mitosis but continue with endomitosis.

In specialty laboratories, immunologic probes and flow cytometry are employed to identify megakaryocyte progenitors. Useful flow cytometric progenitor markers are the general stem cell marker CD34, HLA-DR, and platelet glycoprotein IIIa (GP IIb/IIIa, CD41).⁸ Platelet peroxidase, localized in the endoplasmic reticulum of progenitors and megakaryoblasts, also may be identified by cytochemical stain in transmission electron microscopy. Identical peroxidase activity is localized to the dense tubular system of mature platelets.⁹

Terminal Megakaryocyte Differentiation

Megakaryocyte progenitors leave the proliferative phase and enter terminal differentiation, a series of stages in which microscopists begin to recognize their unique Wright-stained morphology in bone marrow aspirate films (Figure 13-3) or hematoxylin and eosin–stained bone marrow biopsy sections (Table 13-1).

TABLE 13-1

Features of the Three Terminal Megakaryocyte Differentiation Stages

	MK-I	MK-II	MK-III
% Precursors	20	25	55
Diameter	14-18 µm	15-40 µm	30-50 µm
Nucleus	Round	Indented	Multilobed
Nucleoli	2-6	Variable	Not visible
Chromatin	Homogeneous	Condensed	Deeply but variably condensed
Nucleus-to-cytoplasm ratio	3 : 1	1 : 2	1 : 4
Mitosis	Absent	Absent	Absent
Endomitosis	Present	Ends	Absent
Cytoplasm	Basophilic	Basophilic and granular	Eosinophilic and granular
α-granules	Present	Present	Present
δ-granules	Present	Present	Present
Demarcation system	Present	Present	Present

MK-I, Megakaryoblast; MK-II, promegakaryocyte; MK-III, megakaryocyte.

Morphologists call the least differentiated megakaryocyte precursor the MK-I stage or megakaryoblast. Although they no longer look like small lymphocytes, megakaryoblasts cannot be reliably distinguished from myeloblasts or pronormoblasts using light microscopy (Figure 13-4). The morphologist may see plasma membrane blebs, blunt projections from the margin that resemble platelets. At this stage the megakaryocyte begins to develop most of its cytoplasmic ultrastructure, including procoagulant-laden α-granules, δ-granules (dense bodies), and the demarcation system (DMS).¹⁰

The later section on mature platelet ultrastructure discusses α-granules and δ-granules with their contents. The DMS is a series of membrane-lined channels that invade from the plasma membrane and grow over the course of terminal differentiation to subdivide the entire cytoplasm. The DMS is biologically identical to the plasma membrane and ultimately delineates the individual platelets during thrombocytopoiesis.

If there is a clinical need, immunologic probes or cytochemical markers are employed for definitive identification by flow cytometry (Table 13-2). In addition to the progenitor markers listed previously, the megakaryoblast may be identified using immunologic probes for the more differentiated membrane structures GP Ib, which is part of the GP Ib/IX/V von Willebrand factor (VWF) adhesion receptor (CD42); mpl, which is the TPO receptor site; or cytoplasmic VWF, detected by histochemical immunostaining rather than flow cytometry. CD36, which is GP IV, becomes visible in the developing MK-II stage, and fibrinogen may be detected by immunostaining in the fully developed megakaryocyte.

TABLE 13-2

Immunologic Probe or Cytochemical Markers at Each Stage of Megakaryocyte and Platelet Maturation

BFU-Meg	CFU-Meg	LD-CFU-Meg	MK-I	MK-II	MK-III	Platelets
CD34; stem cell marker
	HLA-DR
	Peroxidase by cytochemical stain in transmission electron microscopy (CD41) glycoprotein IIb/IIIa (fibrinogen receptor) by flow cytometry
			CD42; glycoprotein Ib, part of VWF receptor, by flow cytometry mpl (TPO receptor site) by flow cytometry PF4 by flow cytometry VWF by immunostaining

  CD36; glycoprotein IV   Fibrinogen

BFU-Meg, Burst-forming unit–megakaryocyte; CFU-Meg, colony-forming unit–megakaryocyte; LD-CFU-Meg, low-density colony-forming unit–megakaryocyte; MK-I, megakaryoblast; MK-II, promegakaryocyte; MK-III, megakaryocyte; PF4, platelet factor 4; TPO, thrombopoietin; VWF, von Willebrand factor.

Thrombocytopoiesis (Platelet Shedding)

Figure 13-7 appears to show platelet shedding. One cannot find reliable evidence for platelet budding or shedding simply by examining megakaryocytes in situ, even in well-structured bone marrow biopsy preparations. In megakaryocyte cultures examined by transmission electron microscopy, the DMS dilates, longitudinal bundles of tubules form, cytoplasmic extensions called proplatelet processes develop, and transverse constrictions appear throughout the processes.¹² The proplatelet processes pierce through or between sinusoid-lining endothelial cells, extend into the venous blood, and release platelets (Figure 13-8). Sometimes whole megakaryocytes escape the marrow in this fashion to lodge in other organs, such as the lungs. Morphologists presume that thrombopoiesis leaves behind naked megakaryocyte nuclei to be consumed by marrow macrophages, although these are rarely seen in bone marrow aspirate films. Their absence leads a few morphologists to speculate that the lung is the primary site of thrombopoiesis.¹³

Hormones and Cytokines of Megakaryocytopoiesis

TPO is a 70-kD molecule with 23% homology with erythropoietin.¹⁴ Messenger ribonucleic acid (RNA) for TPO has been found in the kidney, liver, stromal cells, and smooth muscle cells, though liver has the most copies. TPO circulates in plasma and is the ligand that binds a megakaryocyte and platelet membrane receptor protein, mpl, named for v-mpl, a viral oncogene associated with murine myeloproliferative leukemia. The concentration of TPO is inversely proportional to platelet and megakaryocyte mass, which implies that membrane binding and consequent disposal of TPO by platelets is the primary control mechanism.¹⁵ Investigators have used in vitro and in vivo experiments to show that TPO induces stem cells to differentiate into megakaryocyte progenitors in synergy with cytokines and that it further induces differentiation of megakaryocyte progenitors into megakaryocytes, induces the proliferation and maturation of megakaryocytes, and induces platelet release (Table 13-3).^16,17 Recombinant TPO in several forms elevates the platelet count in healthy donors and in patients treated for a variety of neoplasms, including acute leukemia, and the commercial form NPlate (romiplostim, Amgen) is effective in raising the platelet count in immune thrombocytopenic purpura.¹⁸

Cell-derived stimulators of megakaryocytopoiesis include interleukin-3 (IL-3), IL-6, and IL-11. IL-3 seems to act in synergy with TPO to induce early differentiation of stem cells, whereas IL-6 and IL-11 act in the presence of TPO to enhance the later phenomena of endomitosis, megakaryocyte maturation, and platelet release. IL-11 has been synthesized and marketed by Genetics Institute as Neumega (oprelvekin) and used to stimulate platelet production in patients with chemotherapy-induced thrombocytopenia.¹⁹ Other cytokines and hormones that participate synergistically with TPO and the interleukins are stem cell factor, also called kit ligand or mast cell growth factor; granulocyte-macrophage colony-stimulating factor (GM-CSF); granulocyte colony-stimulating factor (G-CSF); and erythropoietin (EPO). The list continues to grow.

Platelet factor 4 (PF4), β-thromboglobulin, neutrophil-activating peptide 2, IL-8, and other factors inhibit in vitro megakaryocyte growth, which indicates that they may have a role in the control of megakaryocytopoiesis in vivo. Internally, reduction in the transcription factors FOG, GATA-1, and NF-E2 diminish megakaryocytopoiesis at the progenitor, endomitosis, and terminal maturation phases.²⁰

Platelets

The proplatelet process sheds platelets, cells consisting of granular cytoplasm with a membrane but no nuclear material, into the venous sinus. Their diameter in the monolayer of a Wright-stained peripheral blood wedge film averages 2.5 µm. MPV, as measured in an isotonic suspension flowing through the detector cell of a clinical profiling instrument, ranges from 8 to 10 fL. A frequency distribution of platelet volume is log-normal, however, which indicates a subpopulation of large platelets (see Figure 39-14). Volume heterogeneity in normal healthy humans reflects variation in platelet release volume and is not a function of platelet age or vitality, as many authors have previously assumed.⁵

Circulating, resting platelets are biconvex, although collection of blood using the anticoagulant ethylenediaminetetraacetic acid (EDTA) induces them to “round up.” On a Wright-stained wedge-preparation blood film, platelets appear circular to irregular, lavender, and granular, although their small size makes them hard to examine for internal structure. In the blood, their surface is even, and they flow smoothly through veins, arteries, and capillaries. In contrast to leukocytes, which tend to roll along the vascular endothelium, platelets cluster with the erythrocytes near the center of the blood vessel. Unlike erythrocytes, however, platelets move laterally with the leukocytes into the white pulp of the spleen, where both become sequestered.

The normal peripheral blood platelet count is 150 to 400 × 10⁹/L. This count represents only two thirds of available platelets, because the spleen sequesters an additional one third. Sequestered platelets are immediately available in times of demand, for example, in acute inflammation or after an injury, major surgery, or plateletpheresis. In hypersplenism or splenomegaly, increased sequestration may cause a relative thrombocytopenia. Under conditions of hemostatic need, platelets answer cellular and humoral stimuli by becoming irregular and sticky, extending pseudopods, and adhering to neighboring structures or aggregating with each other.

Reticulated platelets, sometimes known as stress platelets, appear in compensation for thrombocytopenia.²¹ Reticulated platelets are markedly larger than ordinary mature circulating platelets; their diameter in blood films exceeds 6 µm and their MPV reaches 12 to 14 fL.²² Like ordinary platelets, they round up in EDTA, but in citrated whole blood, reticulated platelets are cylindrical and beaded, resembling megakaryocyte proplatelet processes. Reticulated platelets carry free ribosomes and fragments of rough endoplasmic reticulum, analogous to red blood cell reticulocytes, which has triggered speculation that they arise from early and rapid proplatelet extension and release. Nucleic acid dyes such as thiazole orange bind the RNA of the endoplasmic reticulum. This property is exploited by profiling instruments to provide a quantitative evaluation of reticulated platelet production under stress, a measurement that may be more useful than the MPV.²³ This measurement is confounded by platelet dense granules, which falsely raise the reticulated platelet count by taking up nucleic acid dyes.

Platelet Ultrastructure

Platelets, although anucleate, are strikingly complex and are metabolically active. Their ultrastructure has been studied using scanning and transmission electron microscopy, flow cytometry, and molecular sequencing.

Resting Platelet Plasma Membrane

The platelet plasma membrane resembles any biologic membrane: a bilayer composed of proteins and lipids, as diagrammed in Figure 13-9. The predominant lipids are phospholipids, which form the basic structure, and cholesterol, which distributes asymmetrically throughout the phospholipids. The phospholipids form a bilayer with their polar heads oriented toward aqueous environments—toward the plasma externally and the cytoplasm internally. Their fatty acid chains, esterified to carbons 1 and 2 of the phospholipid triglyceride backbone, orient toward each other, perpendicular to the plane of the membrane, to form a hydrophobic barrier sandwiched within the hydrophilic layers.

Buy Membership for Hematology, Oncology and Palliative Medicine Category to continue reading. Learn more here