David W. Sammons

Fetal cells in maternal blood: recovery by charge flow separation

Fetal blood cells can be recovered from the maternal circulation by charge flow separation (CFS), a method that obviates the risks associated with amniocentesis and chorionic villus sampling. By CFS, we processed blood samples from 13 women carrying male fetuses, 2 carrying fetuses with trisomy 21, and 1 who had delivered a stillborn infant with trisomy 18. On average more than 2000 fetal nucleated red blood cells were recovered per 20-ml sample of maternal blood. Recovery of fetal cells was confirmed by fluorescence in situ hybridization with probes for chromosomes Y, 18 and 21. After culturing of CFS-processed cells, amplification by the polymerase chain reaction revealed Y-chromosomal DNA in clones from four of six women bearing male fetuses, but not in clones from three women bearing female fetuses.

Charge flow separation: quantification of nucleated red blood cells in maternal blood during pregnancy.

We set out to ascertain the numbers of fetal cells that enter the maternal blood stream during pregnancy. Samples of 15–16 ml of whole blood were collected from 225 women—mostly 10–18 weeks pregnant—and then processed by charge flow separation, a novel method based on free flow electrophoresis in a buffer counterflow gradient. After their recovery in four different separation instruments, nucleated red blood cells (NRBC) were enumerated histologically. In some cases fetal NRBC were identified and enumerated by fluorescence in situ hybridization with probes for the X and Y chromosomes and fetal haemoglobin mRNA. Recoveries were consistent among the four separation instruments: the median numbers of NRBC obtained were 4190, 1590, 2805 and 3860. Our data show that approximately 30 per cent of those cells were fetal. Thus, recent reports on the separation of fetal NRBC by other methods, give underestimates of their frequency in maternal blood.

Fetal cells in maternal blood

Fetal lymphocytes, trophoblasts, and nucleated red blood cells have each been separated from maternal blood by methods such as flow cytometry, magnetic cell sorting, and charge flow separation. The frequency of fetal cells among circulating maternal mononuclear cells remains to be ascertained. Current estimates range from about 10−5 to 10−7, but the numbers may be increased in women carrying aneuploid fetuses. Fetal cells separated from maternal blood have been studied by methods such as polymerase chain reaction and fluorescence in situ hybridization. Among fetal conditions so far identified are sex; human leukocyte antigen and Rh blood types; trisomy 13, 18 and 21; triploidy; and sickle cell anemia and thalassemia. Thus, fetal cell separation might one day be used for screening of the common aneuploidies and, ultimately, for prenatal diagnosis. Individual fetal erythroid precursors have been cultured after separation in some laboratories. Culturing and karyotyping of separated fetal cells might enable diagnosis of a spectrum of chromosomal and genetic disorders. Further development will be required, however, before regular clinical application of these methodologies.

The diagnostic histopathologic features of ocular malaria.

BACKGROUND The ocular complications in patients with malaria have been studied clinically by many investigators, but the histopathologic changes were rarely described and generally regarded as nonspecific. METHODS The eye of a 53-year-old man who died of chloroquine-resistant Plasmodium falciparum malaria was studied by brightfield and polarized light microscopy. FINDINGS An epibulbar hemorrhage that involved the conjunctiva, episclera, and tendinous insertion of the medial rectus muscle was present. Cytoadherence and rosetting of the parasitized erythrocytes were observed within the partially occluded lumens of small retinal and uveal blood vessels. The birefringence of hemozoin (malarial pigment) within the lumens of small ocular blood vessels and in the hemorrhagic epibulbar area was demonstrated by polarized light. CONCLUSION Birefringent hemozoinemia in vascular lumens of ocular tissues indicates systemic malarial infestation by any of the four species of malaria. Cytoadherence and rosetting of the parasitized erythrocytes inside ocular capillaries and venules is diagnostic of P. falciparum and is an important cause of ocular hemorrhage.

Frequency of nucleated red blood cells in maternal blood during the different gestational ages

We wished to determine the time of pregnancy at which optimal numbers of nucleated red blood cells (NRBC) are present in maternal blood. Because 30% of the NRBC in maternal blood are fetal, there are implications for prenatal screening and diagnosis. Samples of whole blood were collected from each of 225 women at various times during pregnancy. The samples were processed by charge flow separation (CFS), the NRBC enumerated, and the numbers compared on a week-to-week basis. To quantify the relationship between week of pregnancy and actual and log-transformed numbers of NRBC recovered, Pearson product moment and Spearman correlation coefficient were estimated for each of four CFS instruments and for the four instruments combined. When the data were analyzed, we found no relationship between stage of pregnancy and numbers of NRBC recovered. Even after logarithmic transformation, variability among the women, estimated by standard deviation, was large and relatively stable across the different stages of pregnancy. The number of NRBC recoverable by CFS appears to be constant between 7 and 25 weeks.

Isolation by preparative isoelectric focusing of a direct acting fibrinolytic enzyme from the venom of Agkistrodon contortrix contortrix (southern copperhead)

Fibrolase, a blood clot-lysing enzyme, was isolated from the venom of the snake Agkistrodon contortrix contortrix using preparative scale isoelectric focusing in the recycling isoelectric focusing (RIEF) apparatus. Two sequential purifications, beginning with 1.0 g of whole, dried venom, were employed. A pH 6-8 range gradient effected the first separation. While 100% of the enzyme was recovered in three fractions, 43% (one fraction) had 70% purity. The second run was a refractionation of three, pooled fractions from the first run, in a 0.7 pH range gradient. Of the fibrolase in the venom, 63% was recovered in four fractions. One of these represented 29% of venom fibrolase, with 97% purity. Gel filtration chromatography removed most of the remaining, higher molecular weight contaminants of the RIEF-purified enzyme.

Isolation by preparative free-flow electrophoresis and aqueous two-phase partition from rat adipocytes of an insulin-responsive small vesicle fraction with glucose transport activity

Preparative free-flow electrophoresis and aqueous two-phase polymer partition were used to obtain a plasma membrane-enriched fraction of adipocytes isolated from epididymal fat pads of the rat together with a fraction enriched in small vesicles with plasma membrane characteristics (thick membranes, clear dark-light-dark pattern). The electrophoretic mobility of the small vesicles was much less than that of the plasma membrane consistent with an inside-out orientation whereby charged molecules normally directed to the cell surface were on the inside. When plasma membranes and the small vesicle fraction were isolated from fat cells treated or not treated with 100 microU/ml insulin and the resident proteins of the two fractions analyzed by SDS-PAGE, the two fractions exhibited characteristic responses involving specific protein bands. Insulin treatment for 2 min resulted in the loss of a 90 kDa band from the plasma membrane. At the same time, a ca. 55-kDa peptide band that was enhanced in the plasma membrane was lost from the small vesicle fraction. The latter corresponded on Western blots to the GLUT-4 glucose transporter. Thus, we suggest that the small vesicle fraction with characteristics of inside-out plasma membrane vesicles may represent the internal vesicular pool of plasma membrane subject to modulation by treatment of adipocytes with insulin.