Mature micromegakaryocytes: an unusual developmental pattern in term infants.

We sought to gain perspective on platelet production in the fetus and the newborn by counting and characterizing megakaryocytes from available cord blood. Elutriation was used to isolate circulating megakaryocytes from umbilical arteries and veins obtained at scheduled caesarean sections of nine normal term fetuses. Megakaryocytes were identified by established criteria, their diameters measured, and maturation stages recorded. Large numbers of megakaryocytes, mostly mature, were found in both the umbilical arteries and veins, many times more than previously observed circulating in adult blood. In term infants more than a third of the mature megakaryocytes had unusually decreased nuclear lobation and were dwarf cells with diameters as small as 13 microns, which we considered to be micromegakaryocytes. The atypicality of these small but mature cells is seen as merely a leftward skewing in the development of megakaryocyte ploidies. We believe that in normal fetuses the extent of megakaryocyte ploidization and development is distinctive and probably regulated differently to the adult pattern.

Circulating megakaryocytes: delivery of large numbers of intact, mature megakaryocytes to the lungs.

To determine the locus of platelet production, we sought to determine if sufficient megakaryocytes reach the lungs in a state that could produce platelets. Elutriation was used to isolate megakaryocytes from blood reaching and leaving the lungs of 20 patients undergoing routine cardiac catheterizations. A mean of 5.0 intact megakaryocytes/ml were found in pulmonary artery blood, compared to only 0.5 megakaryocytes/ml, with partial cytoplasmic content, in aortic samples. The megakaryocytes in central venous and aortic samples were all mature. The identity of these cells as megakaryocytes, their maturity and normal morphology were confirmed by standard and immunoelectron microscopy. Cardiac outputs were obtained for each patient at the time of blood sampling, allowing an extrapolation that 40 x 10(6) intact, mature megakaryocytes were being delivered to the lungs every day in the average patient, compared to only 4.0 x 10(6) partially spent megakaryocytes exiting the lungs daily. About 98% of megakaryocyte cytoplasm reaching the lungs did not exit as recognizable megakaryocytes or fragments. The number and state of the megakaryocytes apparently filtered in the lungs is consistent with the hypothesis that megakaryocytes may shed platelets within the pulmonary microvasculature, which may be the primary site of platelet production.

Elutriation for isolation of megakaryocytes.

Successful isolation of guinea pig megakaryocytes in large numbers was first achieved with a combination of techniques, taking sequential advantage of the low relative densities and large diameters of most megakaryocytes. Several laboratories have made minor improvements, but this approach retains the disadvantage of losing a significant fraction of the megakaryocyte population, the small immature ones. Counterflow centrifugal elutriation has been shown to eject cells from a chamber progressively, according to their sizes. Because almost all the megakaryocytes are bigger than the other marrow cells, the megakaryocytes can be retained while rejecting the contaminants. With this technology, yields of 1.4-2.0 x 10(6) megakaryocytes from one guinea pig are routine, recoveries have been 93%-94% of the input number of megakaryocytes, and final purities now average 72%. A split-specimen comparison with our previous method found elutriation to provide much greater yield and recovery with at least as great a purification as the density-velocity combination. This new technique was easily adapted to isolation of megakaryocytes in single aspirates from normal human marrow. Fifty-fold purification with near total recovery and a yield of 27,000 megakaryocytes per donor allows easy and reliable cytologic studies. Elutriation appears to be the current method of choice for isolation of megakaryocytes.

Cellular retinyl esters and retinol among parenchymal and stellate cells in normal rat liver.

[11,12-3H] Retinyl acetate (100 micrograms/20 microCi/rat) in corn oil was fed by stomach tube to normal male Wistar-Furth rats (approximately 250 g body weight). After 15 days, the contents of retinyl esters and retinol (total retinol) and their 3H-radioactivity were measured in the whole liver, crude parenchymal cells and the purified parenchymal cells, employing differential centrifugation, centrifugal elutriation and high performance liquid chromatography (HPLC) techniques. Of the total liver retinol (nmol/g liver), the crude parenchymal cells had nearly 90%, whereas the purified parenchymal cells had only 21% based on HPLC analysis. Furthermore, of the total liver retinol radioactivity (dpm/g liver) the crude parenchymal cell fraction had 85%, while the purified parenchymal cell fraction had only 16%. Based on the cell number, the crude parenchymal cell fraction was contaminated by retinoid-rich stellate cells to the extent of 4%. It, therefore, was concluded that the parenchymal cells accounted for 16-21%, whereas the stellate cells contributed 79-84% of total retinol stored in the liver under normal steady-state conditions. It also was calculated that on a per mg basis, stellate cells had 200 times more total retinol than parenchymal cells, whereas on a per cell basis each stellate cell had 74 times more total retinol than a parenchymal cell.