Observations on plasma and red cell lipids in hereditary spherocytosis

Plasma lipids and red cell lipids were determined in hereditary spherocytosis (HS), 15 unsplenectomized and eight splenectomized patients. Plasma lipids (cholesterol: total, free and high density lipoproteins, free fatty acids, and phospholipids) were markedly decreased in HS, especially in the unsplenectomized patients. Concomitantly, red cell membrane lipids (free cholesterol and phospholipids: total, phosphatidyl ethanolamine, sphingomyelin, phosphatidyl choline, and lysophosphatidyl choline) were also diminished. The plasma lipids stay low even after splenectomy although partially restored. Thus, decreased membrane surface, a hallmark of HS, could be partly attributed to the diminished membrane lipids induced by plasma lipid abnormalities in the patients.     

Effect of long-term physical training on total red cell volume

Six months of military training elicited a significant 4% mean increase in total red cell volume (TRC) measured by the 51Cr-labelled red cell method. This increase was associated with a mean 16% increase in predicted maximal oxygen uptake (VO2max). The increases in TRCV and predicted VO2max were inversely related to their initial levels. A statistically highly significant correlation between TRCV and predicted VO2max was observed (r = 0.59). The trained group had larger initial TRCVs than the sedentary group and the subjects who became well conditioned had a significant increase in TRCV, which contrasted with the unchanged TRCV in the subjects who did not become well conditioned. The greatest increase in TRCV (11%) was found in corporals, who had the hardest training. A statistically significant correlation between the changes in TRCV and estimated plasma volume was observed (r = 0.62), P less than 0.001). Owing the 1.8% increase in body weight the TRCV and predicted VO2max in terms relative to the body weight did not demonstrate the changes as clearly as did the absolute values. The factors affecting the TRCV increase are discussed.     

Sensitivity of column agglutination technology in detecting unexpected red cell antibodies

Summary. This study was conducted to compare a new microtube column agglutination technology (CAT) with a previously described tube saline-indirect antiglobulin test (SIAT) with carefully defined performance in the detection of unexpected antibodies. On testing 117 sera from fresh and frozen stock containing antibodies detectable by SIAT, CAT failed to detect two examples of weak anti-K. All other discrepancies between the two techniques involved antibodies generally regarded as clinically insignificant. Titration studies with anti-D (concentration approximately 10 ng/ml) and 23 other antibodies, and studies with 10 weak antibodies of various blood group systems, showed the two techniques to be of similar sensitivity. Equivocal CAT results requiring repeated testing were found in 4·1% of specimens tested. We conclude that the CAT method adequately meets our requirements in sensitivity of detection of unexpected antibodies in pre-transfusion testing and offers opportunities for savings in technical staff time.     

Altered red cell and platelet adhesion in hemolytic diseases: Hereditary spherocytosis,paroxysmal nocturnal hemoglobinuria and sickle cell disease

Objectives

Intravascular hemolysis may have important pathophysiological consequences, such as the induction of cellular adhesion and vasculopathy. We compared the adhesive properties of red cells (RBC) and platelets in hereditary spherocytosis (HS), paroxysmal nocturnal hemoglobinuria (PNH) and sickle cell disease (SCD) patients.

Design and methods

The adhesion of RBC and platelets, from patients and healthy subjects, was determined using static adhesion assays. RBC surface markers were characterized by flow cytometry and lactate dehydrogenase (LDH), plasma hemoglobin (pHb) and TNF-α were assayed in serum/plasma samples.

Results

pHb levels were elevated in all three hemolytic diseases, indicating the incidence of intravascular hemolysis. RBC adhesion and TNF-α were augmented in HS and SCD, but not in PNH. Reticulocyte counts were raised in the three diseases, but were higher in HS and SCD than in PNH; high expressions of CD71, CD36 and CD49d were observed on SCD RBC, while CD71 alone was increased on HS and PNH RBC. Splenectomy was associated with reversals of increased pHb, RBC adhesion, reticulocytes, RBC marker expression and inflammation in HS. In contrast, platelet adhesion was elevated in SCD and PNH, but not HS. Platelet adhesion correlated significantly with serum LDH, but not pHb, in the hemolytic disease cohort; interestingly, LDH did not correlate with reticulocytes or pHb levels.

Conclusions

Results indicate that extravascular, rather than intravascular, hemolysis (and ensuing RBC production) may contribute to elevations in RBC adhesive properties in HS and SCD, while mechanisms peculiar to each disease may augment platelet adhesion in SCD and PNH.     

The use of pooled red cells and column techniques for routine red cell antibody detection

Summary. The object of antibody screening is to detect all clinically relevant antibodies. In order to do this effectively red cells are selected with an appropriate antigen profile. The introduction of column techniques for antibody screening by indirect antiglobulin testing (IAT) and two-stage enzyme testing (ETC) is perceived to lead to an increased sensitivity and an ability to detect red cell antibodies more easily than by traditional tube techniques because reactions in columns are more easily read and are stable. We evaluated the use of a column technology with pooled red cells for routine antenatal screening. The pooled cells used contained at least one cell with homozygous antigen expression for the majority of clinically significant antibodies known to be present, except for Kell. Pooled cell results were not as easy to read in gel columns when compared with single cell results due to weaker reactions which were often diffused throughout the gel in the column. We concluded that the use of pooled cells led to a decreased sensitivity which proved problematic for the interpretation of results. We used a two-cell and a three-cell pool and found that detection of known antibodies was reduced in IAT and ETC methods.     

Reduced volume of red blood cell priming is safe for pediatric patients undergoing therapeutic plasma exchange

RationalTherapeutic Plasma Exchange (TPE) procedures in pediatric patients are challenging due to the large extracorporeal volume of the cell separators, which were designed for adults. Red blood cell (RBC) priming is an alternative for overpassing the risks of hypovolemia, but data referring to the volume of packed RBCs to be infused are yet incomplete. Restricting the volume of RBC priming may potentially be associated with less transfusion reactions.GoalTo determine the safety of administering a reduced volume of RBC priming for pediatric patients undergoing TPE, in comparison to the standard volume recommended by the cell separators’ manufacturers.MethodsThis was a case-control study which enrolled 15 pediatric patients undergoing TPE and weighting more than 10Kg. The TPE procedures (n = 406) were divided in two groups: 1) Group1: TPE with ≤150 mL of packed RBC priming and 2) Group2: TPE with 150-250 mL of RBC priming. Groups were compared in terms of hemoglobin / hematocrit and occurrence of adverse reactions.ResultsGroup1 and Group2 did not differ significantly in relation to pre- and post-TPE hemoglobin (Hb) levels (p = 0.19 and p = 0.18, respectively). The Δ Hb (Hb pre-TPE – Hb post-TPE) was also not statistically different between the groups. The number of adverse reactions was significantly higher in Group 2 in relation to Group 1 (p = 0.01). The number of allergic reactions was also higher in Group 2 (p = 0.06).ConclusionsRestricting the volume of RBC priming to less than 150 mL is safe for pediatric patients weighting more than 10Kg and associated with lower rates of transfusion-related adverse reactions.     

Abnormal membrane protein of red blood cells in hereditary spherocytosis

We present evidence that the hereditable hemolytic disease, hereditary spherocytosis (HS), involves an abnormality in protein of the red cell membrane. Unlike that from normal red cells, lipid-free proteins extracted from HS red cell membranes fail to increase in sedimentation rate when treated with cations; such treatment of normal membrane proteins has been shown by others to cause the formation of microfilaments. That microfilament formation might be defective in HS red cell membranes is supported by observations with vinblastine. This compound, a potent precipitant of filamentous, structure proteins throughout phylogeny, precipitates significantly less HS membrane protein than normal. The resistance of HS membrane protein to changes in conformation by cations is observable at the cellular level as well. That is, both normal and HS red cells agglutinate after repeated washing and suspension in electrolyte-free media. Tiny concentrations of Ca⁺⁺ (5 × 10^-5 M) changes the surfaces of normal cells in such a way as to cause disagglutination; HS red cells resist this change and remain agglutinated unless Ca⁺⁺ concentrations are increased many-fold.