Frank H. Gardner

Chromosome studies in "preleukemia". III. Myelofibrosis.

Cytogenetic studies were done on 18 patients with myelofibrosis or the closely related syndrome, undifferentiated myeloproliferative disorder (MPD). Clones of cells with chromosome abnormalities were demonstrated in the blood of eight individuals, including two with a history of radiation therapy and two with “acute myelofibrosis.” Trisomy 8 was present in the latter two patients, but otherwise, there was no consistent cytogenetic pattern or correlation with specific hematologic findings. Sixteen of these patients have been followed for more than 1 year or until death; none has progressed to leukemia. The results indicate that chromosome abnormalities are relatively common in this disorder, but as with polycythemia vera, and unlike some other “preleukemic” states, the aberrant clones in myelofibrosis do not appear to indicate that clinical leukemia is imminent.

Two complex translocations in chronic granulocytic leukemia involving chromosomes 22, 9, and a third chromosome

SummaryAmong 13 Ph-positive cases of chronic granulocytic leukemia (CGL), banding studies revealed two with complex rearrangements involving translocation of the long arm of number 22 to another autosome and a segment of that chromosome translocated to the long arm of number 9. In a patient with both CGL and sickle cell anemia, the 3-way rearrangement involved chromosomes 5, 9, and 22; and he also had a second Phildaelphia chromosome and two constitutional variants: pericentric inversion of the other number 9 chromosome and satellite polymorphism in the G group. The karyotype of the leukemic cells was interpreted as: 47,XY,inv(9) (p11q13),t(5;9;22)(q13;q34;q11)+del(22)(q11).In the second patient, the complex translocation in the Ph-positive cells involved chromosomes 3, 9, and 22, resulting in a karyotype interpreted as: 46,XX,t(3;9;22)(p21;q34;q11).Several reports indicate that an abnormality of chromosome 9 is not essential for the development of Ph-positive CGL, but the very high frequency of its involvement (including these unusual translocations) suggests that some type of non-random somatic association may exist between 9q and 22q which makes simultaneous breakage likely.Attempts to correlate specific types of Ph chromosome rearrangements with the clinical course of CGL must await the identification of more cases and longer follow-up.

Mapping human autosomes: evidence supporting assignment of rhesus to the short arm of chromosome No. 1.

Rh-negative erythrocytes were found in the blood of an Rh-positive man suffering from myelofibrosis. Nucleated hemopoietic precursors were also circulating in his blood, and these cells had an abnormal chromosome complement from which identifiable chromosome segments had been deleted. Correlation of the serological and cytogenetic findings, combined with previous data, indicates that the Rhesus blood group locus is on the distal portion of the short arm of chromosome No. 1.

Platelet Preservation by Freezing. Use of Dimethylsulfoxide as Cryoprotective Agent

Variables important in the preservation of platelets by freezing with dimethylsulfoxide (DMSO) as cryoprotective agent were studied in normal volunteers and thrombocytopenic patients. Use of 5 per cent DMSO and a freezing rate of 1–3 C/min‐ute yielded optimal preservation of platelet viability. The addition of 5 per cent Dextrose did not improve results. In vivo yield using 5 per cent DMSO was superior to previous results in which glycerol was used as the cryoprotective agent. Viability after freezing was equivalent when platelets were frozen in small (10 ml) and large (60 ml) volumes of plasma. The larger volume had the advantage that a smaller percentage of the platelets was lost during transfer from one plastic container to another.

Room Temperature Storage of Platelets

ALTHOUGH OUR GROUP has had a long interest in the techniques for liquid storage of platelets, we have confined our efforts in recent years to an evaluation of the factors which effect the viability of platelets stored at 22 C or room temperature. The modern management of acute leukemia and aplastic anemia requires a platelet preparation which provides not only immediate hemostatic effect but also hemostatic protection for the patient for 48 to 72 hours after infusion. Studies indicate that platelet survival, as measured by 51Chromium, is compromised if platelets are stored at any temperature below 20 C. Such platelets may provide immediate hemostatic benefit but their efficacy beyond the first few hours after infusion is doubtful. On the other hand, storage at temperatures above 24 C is also suboptimal. The major limiting factor in storage a t 22 C is a fall in pH which results from a generation of lactic acid by platelet glycolysis and, to a lesser degree, from the retention of CO, arising from the oxidation of glucose and fatty acids. Viability of platelets is lost if pH falls to 6.0 or below. Since metabolic rate is enhanced at higher temperatures, the range of storage temperature should be 20 to 24 C. Since ambient temperatures in many blood banks may vary outside of this range, it is important to be aware of the narrow range which assures optimal results. Even within this optimal temperature range, other technical factors require careful monitoring. First of all, the concentrates must be agitated. In our experience, failure to agitate leads not only to a more rapid pH fall but also to a reduction in viability of

Biogenesis of erythrocyte membrane proteins in vivo studies in anemic rabbits

To study the process of red cell membrane protein synthesis we have followed the time course of [3-H]leucine appearance in total protein and individual peptides of the erythrocyte membrane following injection of the amino acid into phenylhydrazine-anemic rabbits. Multiple peripheral blood samples were taken from single animals over a 5-week period. Erythrocyte membrane proteins were separated by polyacrylamide gel electrophoresis in sodium dodecylsulfate and dithiothreitol; incorporation of radioactivity was determined by gel slicing and liquid scintillation spectrometry. Appearance of [3-H]leucine in circulating erythrocytes reached a peak at 1-3 days, with a steady decline thereafter. The radioactive amino acid appeared first in the lowest molecular weight peptides and last in the largest peptides; at the earliest time point (8 h), little radioactivity was observed in any of the four largest peptides present in the membranes (bands A, 1, 2 and 3). Certain smaller peptides (bands 4, 5 and 9) were the predominant species labeled at this time. By 24 h all peptides showed significant incorporation. With maturation of the red cells, label largely disappeared from bands A, 9 and several smaller peptides; this was confirmed by finding that the peptides are virtually absent from mature circulating erythrocytes. These data are interpreted as showing that red cell membrane proteins are synthesized asynchronously during the life cycle of the erythrocyte; the largest peptides are made predominantly in the earlier marrow stages of development, while certain of the smaller peptides are still being synthesized in the reticulocyte stage. Several membrane proteins appear to be specific to the reticulocyte and are lost during the process of cell maturation in the circulation.

Red cell maturation: Loss of a reticulocyte-specific membrane protein

Abstract A protein present in red cell ghost membranes from reticulocyterich blood disappears from the membrane in vivo as the reticulocytes mature. This protein, with an estimated molecular weight of 33,000, is not present in mature circulating red blood cells. It is not associated with the ribosomes present in the reticulocyte.

Biogenesis of erythrocyte membrane proteins. In vitro studies with rabbit reticulocytes.

The capability of rabbit reticulocytes to synthesize red cell membrane proteins has been tested in vitro. Reticulocyte-rich blood from phenylhydrazine-treated rabbits was incubated in vitro in a complete amino acid medium containing ferrous salts, glucose, rabbit plasma and [3-H]leucine. Red cell ghost membranes were prepared by hypotonic lysis and leucine incorporation into hemoglobin and total membrane proteins determined. The pattern of incorporation into individual peptides was determined by polyacrylamide gel electrophoresis of labeled membranes on large (19 mm) gels which were then sliced into 1 mm sections; radioactivity was compared with densitometric tracings of Coomassie blue stained analytical (6 mm) gels. Incorporation of [3-H]leucine into both hemoglobin and membrane protein was linear over 1 h. Gel analysis of labeled membranes revealed that the amino acid was primarily incorporated into peptides with molecular weights of 90 000 or less; three peptides of molecular weights 90 000, 60 000 and 33 000 showed the highest specific activity. Synthesis of the four largest peptide species was negligible. Removable of ferrous salts inhibited synthesis of both globin and membrane protein equally (approx. 50%). However, puromycin and cycloheximide preferentially inhibited the synthesis of globin as compared to membrane proteins. Reticulocytes remain capable of synthesizing a number of membrane proteins; these results are consistent with studies of red cell membrane synthesis in anemic rabbits in vivo.

19-Nortestosterone decanoate induced erythropoiesis in the actinomycin-D-treated hyperoxic mouse☆

The erythropoietic effect of an androgen, 19-nortestosterone decanoate (19-ND), on the actinomycin-D- (ACT) treated mouse has been investigated. The toxic effect of a daily administration of 60 μg/kg body weight of ACT on hematopoietic tissue was partially overcome by a daily 1.25 mg injection of the androgen. When an equivalent amount of ACT was administered in fractionalized doses (20 μg/kg body weight at 8-hour intervals), significant levels of erythropoiesis were maintained by identical androgen therapy. These data suggest that 19-ND increases the absolute number of erythroid precursor cells, thus increasing the absolute number of these elements which remain unaffected by the amount of ACT injected. Such unaffected cells will mature to normal erythrocytes, thereby maintaining significant erythropoiesis.

PLASMA HEMOPEXIN AND HAPTOGLOBIN IN HEMOLYTIC DISEASES OF THE NEWBORN

Many factors have been considered in the evaluation of hemolytic diseases of the newborn. At the present time, it may be difficult to estimate the degree of hemolysis accurately, the needs for exchange transfusion and the effect of such a transfusion. Recently it has been shown that the concentration of hemopexin, a B-glycoprotein with high affinity for heme, is decreased in hemolytic diseases in adults and in older children (10, 15). Also the hemopexin level in the normal newborn infant is about 20% of the normal adult level (3). In the present study the plasma hemopexin concentration in a group of normal term and premature infants as well as newborn infants with hemolytic diseases has been determined with an immunodiffusion method in an attempt to assess its value as an aid in the diagnosis of increased red cell destruction. Although it is well known that the haptoglobin level in newborn infants is generally very low (2, 12, 14), immunochemical determinations of haptoglobin were included to evaluate the relationship between these two plasma proteins in the neonatal period.