Robert T. Card

Red cell membrane changes during storage.

A great deal is known about the red cell membrane and its abnormalities in various pathologic states. During red cell storage there is a progressive development of spheroechniocytosis with eventual production of irreversibly nondeformable red cells. The loss of membrane function is most likely related to some abnormality in maintenance of the cytoskeleton of the red cell. These changes appear to occur independent of ATP levels. Despite the increasing knowledge of the structure and function of the red cell membrane very little as yet is known about the specific abnormality in the red cell membrane that occurs during storage in the blood bank. Recent evidence for abnormal spectrin-actin interaction and abnormal spectrin oxidation has been the most promising. Areas of interest for research include studies of the specific mechanisms by which the plasticizer DEHP interacts with the membrane, specific definition of the molecular defect in membrane proteins that occurs during storage, and means to prevent these. If such deterioration and membrane stiffening could be prevented then the quality of the red cells that are transfused would be improved both in their function and ability to survive in the microcirculation. A final need, while not of specific value to the red cell itself, is the development of media and additives that will allow for increased plasma and Factor VIII yields, one of the driving forces in the blood transfusion system.

Biologic and clinical significance of CD7 expression in acute myeloid leukemia.

CD7 antigen, a T‐cell lineage associated antigen, is expressed in a minority of patients with acute myeloid leukemia (AML). The biologic and clinical significance of this finding is not clearly established. In this retrospective study of patients with de novo acute myeloid leukemia, we have identified CD7 expression and analyzed its association with markers expressed early in hemopoietic ontogeny and clinical parameters. Among 60 consecutive AML patients, we found six (10%) expressing CD7 on leukemic cells. There were five males and one female and the mean age was 59.6 years (age range: 32–76 years) with no demographic peculiarities. The FAB subtypes were: M0 (2), M1 (1), M2 (1), and M4 (2). CD7 expression was associated with immature antigens CD34, HLA‐DR, and terminal deoxynucleotidyl transferase (TdT) and antigen receptor gene rearrangements (rearrangements of T‐cell receptor gamma chain in 6/6 and immunoglobulin heavy chain in 2/6). Hepatomegaly was present in three and this was associated with splenomegaly with lymphadenopathy in one patient. Mediastinal or central nervous system involvement was absent. Complete remission was achieved in two patients with standard chemotherapy; one of these is in remission and alive (5 years later), while one died following relapse 9 months later. Three patients had significantly lower response to standard therapeutic regimen (two died during induction and one died 7 months later without ever achieving complete remission). One patient has been excluded in determining the prognostic significance of CD7 due to early death. Our results suggest origin of CD7+ AML from early hemopoietic precursors and indicate biologic aggressiveness in a significant proportion of patients. We suggest evaluation of CD7 in all patients with AML at the time of diagnosis in view of poor clinical outcome. Am. J. Hematol. 58:278–284, 1998.

Cardiovascular disease (CVD) in Canadians with haemophilia: Age‐Related CVD in Haemophilia Epidemiological Research (ARCHER study)

Improvements in haemophilia care have increased life expectancy in persons with haemophilia (PWH). This ageing population presents clinicians with management challenges as they develop age‐related comorbidities such as cardiovascular disease (CVD).

Patterns of tertiary prophylaxis in Canadian adults with severe and moderately severe haemophilia B

From a young age patients with severe and moderately severe FIX deficiency (haemophilia B) can experience spontaneous or traumatic bleeding and joint destruction may result. The use of coagulation factor IX concentrate to prevent anticipated bleeding, as primary or secondary prophylaxis, has become a common and recommended practice in children. The current practice of using tertiary prophylaxis, in the presence of established joint arthropathy, in adults with haemophilia B is not well characterized. This observational study was conducted to gain a better understanding of the recent Canadian experience with tertiary prophylaxis in adults with severe and moderately severe haemophilia B. Data were collected from all eligible adult (≥ 18 years of age) males with baseline FIX:C ≤ 2% from seven Canadian Hemophilia Treatment centres over a 2‐year observation period from 2009 to 2011. Thirty‐four per cent of the 67 subjects with moderately severe haemophilia B were exposed to prophylaxis with the majority as continuous prophylaxis (≥45 weeks year‐1). The severe subgroup (FIX:C < 1%) demonstrated a 52% exposure rate. None had primary prophylaxis exposure in childhood. Eighty‐one per cent used once or twice weekly infusion regimens and reported a median annual bleeding rate of five bleeds per year versus four bleeds per year for those using on‐demand treatment. Annual median factor utilization for all subjects using prophylaxis was 196 283 U year‐1 compared to 46 361 U year‐1 for on demand. Approximately 50% of adults with severe haemophilia B are using continuous tertiary prophylaxis in Canada, a practice likely to increase which warrants further study.

Effects of Blood and Crystalloid Cardioplegia on Cardiac Function at Organ and Cellular Levels During Hypothermic Cardiac Arrest

The present investigation was undertaken to compare the effects of cold crystalloid and blood cardioplegia on the functional recovery of the heart; on Ca+ + binding and uptake, Ca+ + -ATPase of the sarcoplasmic reticulum (SR), and sarcolemmal (SL) ATPase; and on serum MB fraction of creatine kinase (MBCK) after one and a half hours of reperfusion following one hour of ischemic cardiac arrest in dog. This study was made also to determine if the functional changes are related to the changes in biochemistry at the molecular level. The dogs were divided into three groups: sham bypass (SB), cold crystalloid cardioplegia (CC), and pump blood cardioplegia (PB). There was a decrease in the cardiac index (CI), left ventricular work index (LVWI), and mean aortic pressure (MAP) in all three groups. The index of myocardial contractility ((dp/dt)/IIP) and CI were lower in the CC group as compared with the SB and PB groups. All the hemodynamic values for the PB group were similar to those of the SB group except total systemic vascular resistance (TSVR) and left ventricular end-diastolic pressure (LVEDP) which were lower in the PB group. The index of myocardial contractility and cardiac index appeared to be greater in the PB group than in the CC group. There was a decrease in the Ca ++ uptake by SR from both the CC and PB groups. Ca++ binding and Ca++ -ATPase of SR from the PB group were depressed. The sarcolemmal ATPase was unaffected in both groups. The serum MBCK increased in both PB and CC groups, though the increase was smaller in the PB group. These results indicate that the functional recovery of the heart was slightly better with pump blood cardioplegia than with cold crystalloid cardioplegia. The depressed myocardial contractility and cardiac function in the CC group were associated with a decrease in the Ca+ + uptake by SR. However, the decreases in the Ca+ + binding, Ca+ + uptake, and Ca+ + ATPase by SR from the pump blood cardioplegic group were not accompanied by decreases in the cardiac contractility and cardiac function. Myocardial damage as assessed by serum MBCK was smaller in the PB group than in the CC group.

Vitamin E deficiency and erythrocyte deformability in the rat

It has been suggested that erythrocyte deformability is decreased in vitamin E deficiency due to oxidative damage to the cell membrane. Male Wistar rats (66 to 88 g) were fed ad libitum an AIN76-based diet containing tocopherol-stripped corn oil without added vitamin E for 8 weeks (−E; n = 8). Control animals were fed ad libitum the same diet containing 50 IU/kg dl-α-tocopheryl acetate (+E; n = 7). Vitamin E deficiency was confirmed by depressed mean (±SEM) plasma α-tocopherol levels (μmol/L), as measured by high performance liquid chromatography [−E: 0.5 ± 0.1; +E: 20.3 ± 1.8] and elevated hydrogen peroxide-induced hemolysis (%) [−E: 92.6 ± 2.4; +E: 4.2 ± 1.6; P < 0.05 by Students t test]. The only alteration in a complete blood count was a depression in reticulocyte number (× 1012/L) [−E: 0.19 ± 0.02; +E: 0.46 ± 0.03; P < 0.05]. Erythrocyte deformability was measured at standard shear stress under conditions of increasing osmolality in the ektacytometer. Elongation index (the ratio of length to width of the diffraction pattern of the deformed cells) was plotted against osmolality to generate an osmotic deformability profile. EImax (the maximum elongation index) and Ohyper (the osmolality at which the elongation index is half of EImax on the hypertonic arm of the curve) were significantly increased in samples from the −E group (P < 0.05 by Students t test). In summary, erythrocyte deformability as measured by the ektacytometer was not decreased by a subclinical vitamin E deficiency in the rat. In fact, a small but significant increase in maximum deformability was observed in erythrocytes from vitamin E-deficient rats.

Hemophilia management in transfusion medicine

Hemophilia is an X-linked bleeding disorder caused by a deficiency of factor VIII or IX activity. We will review the use of blood products, including plasma derived and recombinant coagulation factor concentrates (CFCs), and other hemostatic agents central to the management of bleeding, surgical procedures, prophylaxis and inhibitors. However, management of hemophilia is more than just giving CFCs. Attention to the physical and psychosocial health of persons with hemophilia to improve their quality of life must be the goal. These goals can best be met by comprehensive care hemophilia (bleeding disorders) clinics designed to provide clinical service and education to persons with hemophilia and their families, and to conduct research to improve hemophilia care.

High 2,3-DPG blood cardioplegia and myocardial preservation during cardiopulmonary bypass.

Blood may provide superior cardioplegia compared with crystalloid cardio plegic solution. However, the results are controversial. This may be due to a leftward shift of the hemoglobin (Hb)-02 dissociation curve induced by hypo thermia, increasing the oxygen affinity for Hb. This effect may negate the po tential benefit of blood cardioplegia. The oxygen affinity for Hb can be decreased by increasing the red cell 2,3-diphosphoglycerate (2,3-DPG), and hence, more oxygen can be delivered to the myocardium. The present investiga tion was undertaken to study the effects of 2,3-DPG-enriched blood cardiople gia on the functional recovery of the myocardium and changes in the coronary sinus red blood cell (RBC) adenosine-triphosphate (ATP), lactate, and RBC DPG after one and a half hours of reperfusion following one hour of ischemic cardiac arrest in dogs. The dogs were divided into three groups: crystalloid (CR); stored blood (SB), and high 2,3-DPG blood (HDPG) cardioplegic groups. Incubation of canine RBC in phosphoenal pyruvate (PEP) led to a 36% increase in DPG and a rightward shift in the Hb-O2 dissociation curve. There was a 4 mm Hg shift in the P50. When compared with the CR group, there was a significant decrease in the cardiac index (CI) and left ventricular work index (LVWI) and a significant increase in the total systemic vascular resistance (TSVR) in the SB group. The CI and LVWI of the HDPG group were similar to those of the CR group, but the TSVR was significantly greater in the former group. The LVWI was significantly greater and the TSVR smaller in the HDPG group as com pared with those in the SB group. The coronary sinus, lactate, and RBC 2,3- DPG were never significantly different at any point among the three groups, although a trend toward a decrease in lactate with the HDPG group was ob served. There was no significant difference in the RBC ATP content from the three different groups except during initial period of ischemic arrest when RBC ATP content of HDPG group was greater than that in CR group. There was a decrease in the Hb content of the blood in all three groups. The decreases were similar in all the groups except during the postpump period, when the Hb con tent of the HDPG and SB groups was higher than that of the CR group. These results suggest that stored blood was inferior to crystalloid and HDPG cardioplegia in preserving the myocardial function and cardiac metabolism. However, HDPG cardioplegia was not better than CR cardioplegia in preserv ing the cardiac function. Better preservation of cardiac function with HDPG as compared with the SB group might be due to a rightward shift in the Hb-O2 dissociation curve.

Erythrocyte deformability in zinc deficiency measured as a function of shear stress in the ektacytometer

Erythrocyte deformability was investigated as a potential biochemical indicator of zinc status. Weanling, male Sprague-Dawley rats were fed ad libitum modified AIN-93G diets containing 3 mg zinc/kg diet (−Zn; n = 10) for 6 weeks. Control rats were pair-fed (+ZnPF; n = 10) or fed ad libitum (+ZnAL; n = 9) diets containing 50 mg zinc/kg diet. Zinc deficiency significantly decreased plasma and tibia zinc concentration (P < 0.001). Erythrocyte deformability was measured on whole blood as a function of shear stress in the ektacytometer. Elongation index, the ratio of length to width of the diffraction pattern of deforming cells, was plotted against shear stress. Maximum elongation index and the initial slope were determined from a function used to fit the sigmoid curve. As analyzed by one-factor analysis of variance, maximum elongation index, a measure of average deformability of the cell population, was significantly depressed in the −Zn and +ZnPF groups as compared with the +ZnAL group (P < 0.05). The mean (±SEM) maximum elongation index for each of the three groups was −Zn, 0.55 ± 0.01; +ZnPF, 0.56 ± 0.01; +ZnAL, 0.59 ± 0.01. The initial slope of the curve, a measure of membrane deformability, was not altered by zinc deficiency. No effect of zinc deficiency was found for deformability of erythrocyte suspensions heated at 48°C for 6 min. Results of this study suggest that erythrocyte deformability would not be a useful functional indicator of zinc status.

Tailored frequency-escalated primary prophylaxis for severe haemophilia A: results of the 16-year Canadian Hemophilia Prophylaxis Study longitudinal cohort

BACKGROUND Severe haemophilia A has high morbidity, and treatment, while effective, is very expensive. We report the 16-year follow-up of the Canadian Hemophilia Prophylaxis Study, which examined the effectiveness of tailored frequency-escalated primary prophylaxis with a focus on health outcomes within the domains of body structures and functions, and activities and participation (according to the WHO International Classification of Functioning, Disability and Health [WHO-ICF] framework) and a view to reducing consumption of costly clotting factor, which accounts for more than 90% of the cost of care of severe haemophilia. METHODS In this longitudinal study, boys with severe haemophilia A from 12 Canadian centres were enrolled at age 1·0-2·5 years. They were treated with standard half-life recombinant factor VIII (SHL-rFVIII), beginning as once-weekly prophylaxis with 50 IU/kg and escalating in frequency (with accompanying dose adjustments) in response to breakthrough bleeding as determined by the protocol. The primary endpoint for this analysis was joint health, as measured by the modified Colorado Child Physical Examination Scores (CCPES) at study end. All analyses were done by intention to treat. The trial is complete, and is registered with ClinicalTrials.gov, number NCT01085344. FINDINGS Between June 26, 1997, and Jan 30, 2007, 56 boys were enrolled. They were followed for a median of 10·2 years (to a maximum of 16·1 years). Median rFVIII usage was about 3600 IU/kg per year. The median end-of-study CCPES physical examination score was 1 (IQR 1-3; range 0-12) for the left ankle and 1 (1-2; 0-12) for the right ankle, with all other joints having a median score of 0. No treatment-related safety events occurred over the duration of the study, including central venous catheter infections. The median annualised index joint bleeding rate was 0·95 per year (IQR 0·44-1·35; range 0·00-13·43), but 17 (30%) patients had protocol-defined unacceptable breakthrough bleeding at some point during the study. INTERPRETATION Tailored frequency-escalated prophylaxis leads to very little arthropathy and very good health outcomes within the WHO-ICF domains, and only uses a moderate amount of expensive clotting factor as compared with standard prophylaxis protocols. Some sequelae of bleeding were observed in our cohort, and future studies should consider a more stringent protocol of escalation. FUNDING This study was initially funded by grants from the Medical Research Council of Canada/Pharmaceutical Manufacturers Association of Canada Partnership Fund and the Bayer/Canadian Blood Services/Hema-Quebec Partnership Fund. Subsequent renewals were funded by Bayer.