Nucleated red blood cells in neonatal medicine

Introduction

It is distinctly uncommon and definitely pathologic to have nucleated red blood cells (NRBCs) in the circulating blood. This statement is true for all humans—in fact, for all mammals—with the singular exception of newborn infants, where small numbers of NRBCs can be a normal finding on the day of birth and perhaps for a day or two thereafter. However, some human newborn infants, particularly those with severe hemolytic disease, severe intrauterine growth restriction, or severe intrauterine hypoxia, have very large numbers of NRBCs in the blood. The explanation for and the implications of this finding are the focuses of this chapter.

Erythroid development in the human fetus

Erythropoiesis is the process whereby RBCs are produced in the bone marrow as well as in the fetal liver of mid- and late-trimester fetuses. The erythropoietic process is a continuous one, but it can be instructive to consider it as occurring in a series of four definable stages: (1) the commitment of pluripotent stem cells into erythroid progenitors, (2) the early erythropoietin-independent stage of clonal expansion of early committed erythroid progenitors, (3) the erythropoietin-dependent late phase involving further clonal expansion and initiation of hemoglobin synthesis, and (4) nuclear condensation, enucleation, and reticulocyte release into the blood.

As shown in Fig. 3.1 , early hematopoietic progenitors, which have the capacity to generate clones of multiple lineages, can be recognized by their capacity to give rise in cell culture systems to colonies that contain mixtures of granulocytes, erythrocytes, macrophages, and megakaryocytes. Colonies containing such a mixture of cell types are derived from a single progenitor cell termed a colony-forming-unit (CFU)–GEMM. Further erythroid commitment of pluripotent hematopoietic progenitors produces a cell capable of generating large clones consisting exclusively of erythrocytes. These progenitors are termed burst forming units–erythroid (BFU-E), which are found primarily in the marrow and fetal liver but also in the circulating blood, where they appear morphologically like an immature blast with a large nucleus containing nucleoli. CFU-GEMM and BFU-E express Epo receptors but at very low densities. BFU-E, after about 14 days in culture, produce colonies containing 500 to perhaps 50,000 hemoglobinized erythroblasts. The early stages of BFU-E proliferation are Epo independent.

The next stage in erythroid development is termed the CFU-E, or colony-forming unit–erythroid. Such progenitors, in culture for 2 or 3 days, generate colonies composed of 8 to about 32 hemoglobinized cells. Most CFU-E are in an active stage of DNA synthesis. Epo receptors are expressed densely on the surface of CFU-E and proerythroblasts. Since those states have the highest density of Epo receptors, they are considered to be the principle hematopoietic targets of Epo and are the most responsive to Epo of any cells. Consequently, high Epo concentrations result in marked expansion of the CFU-E compartment. CFU-E also have a high cell-surface expression of the transferrin receptor (CD 71), as a means of importing iron that will be needed for hemoglobin production required for generating mature erythrocytes.

After the CFU-E stage, maturing erythrocyte precursors can be recognized as morphologically distinct cell types in the marrow and fetal liver ( Fig. 3.2 ). The first is the proerythroblast, a moderate to large oval or round cell with a relatively large nucleus with prominent nucleoli and a rim of basophilic cytoplasm. The next more mature red cell precursor is the basophilic erythroblast, which is slightly smaller with a nucleus that lacks nucleoli. Next, the polychromatic erythroblast is recognized by a mixture of blue and pink cytoplasm (thus polychromatic), the pink color signifying hemoglobin. The nuclear chromatin of that cell type is becoming condensed and smaller. Once the cytoplasm is almost completely pink and the nucleus has become pyknotic and small, the cell is termed an orthochromatic erythroblast. It is at this stage that enucleation occurs, thereby producing a reticulocyte, lacking a nucleus but retaining organelles.

Enucleation of orthochromatic erythroblasts

Circulating RBCs of fish and amphibians have nuclei. Perhaps this enables their erythrocytes to be at least somewhat transcriptionally active and thereby might provide the animals with some survival advantage. However, if that is so, why was erythrocyte enucleation selected for during mammalian evolution? At least three possibilities have been suggested: (1) Enucleation provides more room in erythrocytes for hemoglobin, (2) enucleation likely improves blood circulation by preventing potential flow impedance in capillaries by erythrocytes that have a large and stiff nucleus, thus rendering them less deformable. Deformation and reformation of RBC occurs repeatedly during the circulation when they pass through spaces narrower than 7.5 µm. (3) Reducing blood viscosity by erythrocyte enucleation might reduce cardiac workload.

The mechanisms involved in mammalian erythrocyte enucleation involve several concomitant processes. These include simultaneous nuclear chromatin condensation and increased hemoglobin production. Chromatin condensation is controlled by upregulating histone deacetylases with parallel downregulation of histone transferases. Time-lapse photography of enucleating erythroblasts show that the nucleus migrates to one side of the cytoplasm and lies in close apposition to the erythroblast plasma membrane by a process that involves a ring of actin around the nucleus. The erythroblast submembrane cytoskeletal proteins, such as ankyrin, spectrin, actin, and protein 4.1, move away from the membrane in the area where the nucleus is being extruded. The entire process appears to occur quickly with resealing of the plasma membrane followed by reorganization of the cytoskeletal proteins ( Fig. 3.3 ). Micro RNA species that posttranscriptionally modulate expression of a variety of genes are involved in this intricate process, but clearer definition of the exact processes is needed. The extruded nucleus is termed a pyrenocyte, from Greek pyren , the pit of a stone fruit. Macrophages in the marrow or liver interact with and engulf the pyrenocyte. However, in tissue culture, even in the absence of macrophages, enucleation occurs. Therefore presumably macrophages are not critical to the enucleation process other than to recover the pyrenocyte and recycle its elements.