Mary B. Gibbs

Physical Characteristics of Microaggregates in Stored Blood

The volume of microaggregates 13 to 80 μ in size which developed in blood components stored under varying conditions was measured with a particle size analyzer. The microaggregates were found to develop progressively during storage of ACD whole blood at 4 to 6 C. Coincident with this there was a drop in the platelet count during the first week of storage and a progressive reduction in the absolute granulocyte count. Microaggregate development after storage of various components of ACD blood was proportional to the concentration of platelets and leukocytes prior to storage. The microaggregates settled into the buffy coat after centrifugation and became larger. In vitro studies indicated that they were resistant to dissociation in vitro in comparison to platelet aggregates induced in fresh blood by adenosine diphosphate. Microaggregate formation was greater in CPD than in ACD anticoagulated blood stored at 4 to 6 C for 24 hours, but was not different after seven days of storage. A greater volume of microaggregates was formed in aliquots of ACD blood stored at 4 to 6 C than at room temperature, while no differences were noted after storage of blood in plastic bags or glass vacuum bottles.

Vitamin A in liposomes. Inhibition of complement binding and alteration of membrane structure.

Incorporation of vitamin A aldehyde (retinal) into liposomes had an inhibitory effect on the amount of human complement protein bound in the presence of specific antiserum. The total membrane-bound protein was directly measured on liposomes which were washed after incubation in antiserum and fresh human serum (complement). At every concentration of complement, decreased protein binding was found with liposomes which contained retinal. Binding of the third component of complement (C3) was also measured directly on washed liposomes and was found to be decreased in the presence of retinal. The diminution in protein binding due to retinal was not caused by differences in the amount of antibody bound and this was shown by two experiments. First, specific antibody protein binding to liposomes was directly measured and was essentially unaffected by retinal. Second, liposomes were prepared from lipid extracts of sheep erythrocytes. These liposomes were used as as immunoadsorbants to remove antisheep erythrocyte antibodies. The immunoadsorbant capacity was the same in both the presence and the absence of retinal. A further conclusion from these experiments was that retinal did not change the number of liposomal glycolipid antigen molecules available for antibody binding and thus presumably did not change the total number of lipid molecules present on the outer surface of the liposomes. Retinal did have an effect on the geometric structure of the liposomes. Size distribution measurements were performed in the diameter range of 1-6.35 mum by using an electronic particle size analyzer (Coulter Counter). Liposomes containing retinal were shifted toward smaller sizes and had less total surface area and volume. It was suggested that retinal-containing liposomes may have had a tighter packing of the molecules in the phospholipid bilayer. This effect of retinal on liposomal structure may have been responsible for the observed decreased binding of C3 and total complement protein.

Simple Methods of Storage of Human Erythrocytes in Liquid Nitrogen: Comparative Study of the Agglutinability of Erythrocytes of the ABO Blood Groups Preserved by Various Methods

Small samples of blood for daily serological use were stored in a liquid nitrogen refrigerator either as blood‐sand pellets or on squares of wire screen in specially constructed metal storage‐cylinders. When thawed at 40 C., these samples yielded excellent recoveries of red cells generally with no evidence of hemolysis. Comparison of the agglutinability of red cells representative of the ABO groups by the 50 per cent hemagglutination end‐point assay method showed that the original hemagglutinating activity of the cell was retained 100 per cent after storage frozen in liquid nitrogen for two years and in glycerol for nine months. In contrast to this, a gradual loss of approximately 50 per cent agglutinability was noted with cells from these same donors stored in ACD or DCS (dextrose‐citrate‐sodium chloride) solutions at 10 C. for 33 days.

Immunochemical Studies of the Rh System. IV. Hemagglutination Assay of Antigenic Expression Regulated by Interaction Between Paired Rh Genes

Quantitative hemagglutination assays with anti‐Rh1 (Rh0 or D), anti‐Rh2 (rh′ or C), anti‐Rh3 (rh″ or E) and anti‐Rh4 (hr′ or c) were performed on the erythrocytes of standard donors of known genotype and of members of one family showing segregation and recombination of four complex Rh genes: R1,2,–3,–4, RW1,–2,3,‐4, R−1,–2,‐3,4, and R−1,2,–3,–4. Within the pedigree, duplicate and triplicate examples of each genotype reacted identically. Hemagglutination scores provided concordant data but showed, additionally, that (1) the strength of expression of both Rh1 and Rhw1 (𝓇Rh0 or Du) directly influenced the amount of agglutination obtainable with guinea pig anti‐rhesus serum (anti‐LW), and (2) in this family R−1,–2,–3,5,19 determines more Rh19 reactivity than does R−1,2,–3,5,19 and much more than R1,2,–3,5,w19. The gene R−1,2,–3,–4 was found to depress not only the expression of Rh1 and Rh3 determined by the paired gene, as described previously by others, but Rhw1, Rh2 and Rh4 as well. The gene, R1,2,–3,–4, was also found to depress the Rh3 and Rh4 antigenic expression of a paired gene but not to the same degree. These findings suggest that interaction between paired Rh genes may account for most, if not all, of the quantitative genotype differences previously attributed to single “gene dosage” and to pertinent portions of the so‐called “cis‐trans” effect. In contrast to Rh1 assays, those for Rh2, Rh3 and Rh4 were associated with parallel log‐probit regression lines, a finding suggestive of more homogeneity in regard to specific binding affinities of these antiserums and antigenic determinants.

Quantitative Hemagglutination Inhibition Studies of Blood-group Substances

Quantitative hemagglutination inhibition studies of the pre‐ and postimmune sera of nine human volunteers immunized with hog A substance have confirmed earlier findings that the character of the agglutinins is altered by immunization. Concomitant with immune antihog A antibody production, the slope of the probit‐log hemagglutination inhibition assay curve became steeper, the amount of hog A substance required to inhibit the agglutinins became smaller, and the rate of dissociation of hog‐A‐anti‐A complexes became slower. The observed differences in the reactivativities of these pre‐ and postimmune sera could be attributed to an increase in specificity and firmer binding of the antibodies to the inhibitor substance.

A Simple Manifold Washing Process* for the Preparation of Erythrocytes for the Antiglobulin Test

A simple manifold washing process has been developed for preparing erythrocytes for use in direct and indirect anti‐human globulin tests. The entire process, consisting of 3 washes, is carried out in only 6 minutes without removing the tubes from the centrifuge head. A comparison of results obtained when sensitized red cells are washed three times, either by normal resuspension in tubes full of physiologic saline solution or by manifold washing, demonstrated that the indirect Coombs titration scores were consistently higher for the mechanically washed red blood cells. Sensitized red cells from 6 cord bloods of ABO incompatibilities, which gave negative reactions with manually washed cells, gave distinct positive reactions by the manifold process.