Cord Blood Transplantation Study (COBLT): cord blood bank standard operating procedures.

In 1995, the National Heart Lung and Blood Institute (NHLBI) solicited requests for a proposal (RFP) entitled "Transplant Centers for Clinical Research on Transplantation of Umbilical Cord Stem and Progenitor Cells." Three banks, six transplant centers, and one medical coordinating center (MCC) (Table 1) were funded with the overall goal of banking cord blood units (CBU) using a single manual of operations. Furthermore, the clinical protocols to evaluate the transplant outcome for adult and pediatric recipients of these well-characterized CBU would be analyzed in a uniform fashion. Because of the intense interest of the transplantation community in the policies and procedures for cord blood collection and processing, the principal investigators of the cord blood banks (CBB) and NHLBI elected to submit for publication the rationale and an abridged, but detailed, version of the standard operating procedures (SOP) developed between October 1996 and July 1998 prior to the initiation of the clinical protocols to be performed with these CBU. As the SOP will be refined over time, the complete SOP and subsequent amendments will be published and continually updated on the websites from the MCC-The EMMES Corporation (www.EMMES.com). All forms referred to in this document may be obtained from the EMMES website. It is hoped that the publication of this document will lay down a framework that will not only facilitate the development of other CBB but also help us more rapidly define what constitutes an "acceptable" CBU product.

Safety of the blood supply. Surrogate testing and transmission of hepatitis C in patients after massive transfusion.

OBJECTIVE: To define a risk profile for post-transfusion hepatitis C in patients receiving massive transfusion. SUMMARY BACKGROUND DATA: Hepatitis C accounts for more than 90% of post-transfusion hepatitis. METHODS: Two-hundred twenty-one of 8,765 consecutive trauma admissions to a Level I trauma center received more than 20 units of erythrocytes. Sixty-nine survivors had positive viral serologic tests at least 1 year after transfusion. Surrogate testing for hepatitis C using alanine aminotransferase (ALT) levels and antibodies to hepatitis B core antigen (Core) began in October 1986 and January 1987, respectively. Donor blood for group 1 (pre-ALT/Core) was transfused before surrogate screening was introduced. Donor blood for group 2 (post-ALT/Core) was transfused after surrogate screening. RESULTS: Sixty-nine patients received blood products from 4,987 donors (mean, 72.3 units of exposure). No patient tested positive for antibodies to hepatitis B surface antigen, human immunodeficiency virus, or human T-lymphotrophic virus type 1. However 23.2% tested positive for hepatitis C virus (HCV) as measured by a second-generation enzyme immunoassay (HCV 2.0) and a recombinant immunoblot assay (RIBA), and 21.7% tested positive by HCV 1.0. Antibodies to Core were found in 8.7% of patients. The risk for post-transfusion hepatitis C per unit of exposure is estimated to be 1.52% group 1 (pre-ALT/Core) and 0.239% for group 2 (post-ALT/Core). CONCLUSIONS: The introduction of ALT/Core donor screening by a blood bank reduced the incidence of post-transfusion hepatitis C by 84%. The risk for post-transfusion hepatitis C depends on units of exposure, screening techniques, and prevalence of hepatitis C in the donor population. In our community, the risk for post-transfusion hepatitis C is less than 0.2% per unit of exposure. The population of massively transfused patients may serve as our effective resource for monitoring the safety of the blood supply.

Use of a pneumatic tube system for delivery of blood bank products and specimens.

This study evaluated the effect of pneumatic tube transport on blood bank specimens and products. No important differences were found between aliquots transported in the tube system and those stored in the laboratory as controls. ABO, Rh, antibody detection or identification, direct antiglobulin testing, and elution were studied. Further, no differences in plasma hemoglobin and potassium concentration were found between units of whole blood and packed cells handled in either manner. Platelet counts in platelet concentrates were not decreased and coagulation factor levels in units of fresh-frozen plasma and cryoprecipitate did not decrease after pneumatic transport. The system tested is currently providing expeditious transport of specimens and blood between blood banks and patient care areas.

Loss of blood group A in acute leukemia. Morphologic and biochemical studies of red cells.

A patient with blood type A had acute myelomonocytic leukemia; his red cells (RBCs) typed as O and his serum had anti-B. RBC membranes were isolated from the patient as well as from controls with group A and O red cells. The membranes were incubated with uridine diphosphate (UDP)-N-acetyl-D-14C galactosamine in plasma from the patient and controls with group A and O red cells. RBC membranes from the patient behaved normally in that they incorporated the terminal carbohydrate responsible for blood group A activity. Scanning electron microscopy showed that the patient's RBCs had striking morphologic changes, with marked crenation and numerous knisocytes and dacryocytes. It was concluded that loss of the A antigen in this patient was not due to an abnormality of the enzyme required to convert H substance to A substance. It was postulated that weakening of the A antigen in some patients with leukemia may be related to a steric modification associated with abnormal red cell morphology.

Transformation of human erythrocyte shape by endotoxic lipopolysaccharide.

Human erythrocytes were observed to undergo a discocyte to echinocyte to spheroechinocyte shape transformation during brief incubation with endotoxic lipopolysaccharide. It was concluded that lipopolysaccharide-membrane interactions alter the curvature of erythrocyte membranes.

Storage and survival of red blood cells with elevated sodium levels.

Approximately 25 percent of black blood donors have an elevated red blood cell (RBC) sodium (Nai) level compared with white donors. This elevation results in a significant increase in the mean Nai from black (9.00 +/- 2.96 mmoles/l RBC) as compared to white blood donors (7.04 +/- 1.48 mmoles/l RBC, p less than 0.001). Red blood cells from four black donors with mean Nai levels of 15 +/- 2.8 mmoles/l RBC were stored for 35 days in citrate-phosphate-dextrose-adenine and compared to that of four donors with normal levels of Nai. Serial measurements of red blood cell adenosine triphosphate, diphosphoglycerate, glucose-6-phosphate dehydrogenase, pyruvic kinase, lactate production rates, and intracellular cations showed no differences between the two donor groups. Furthermore, the mean 24-hour posttransfusion survival was not significantly different for the high Nai group (83.2 +/- 5.6%) as compared with the control group (82.3 +/- 6.9%). Based on this study, it is not necessary to eliminate individuals with an elevated red blood cell Nai level as blood donors.

Utilization of a cell separation technique to evaluate patients with a positive direct antiglobulin test.

Density distribution curves of red blood cells (RBC) from patients with a positive direct antiglobulin test (DAT) were compared to a standardized curve constructed from cell column measurements of centrifuged microcapillary tubes filled with RBC and phthalate ester mixtures encompassing a specific gravity range of 1.078 to 1.114. Shortened survival resulted in a loss of older RBC and a shift of the curve to the right over the high specific gravity ester range. Reticulocytosis resulted in a downward shift of the curve over the low specific gravity range. In patients with a positive DAT due to an autoantibody or drug, the density distribution curve was either normal or demonstrated evidence of shortened RBC survival. In patients with a positive DAT due to an alloantibody, however, evidence of shortened survival was not seen. The distribution of antibody on young and old RBC harvested from the appropriate microcapillary tubes depended upon the etiology of that antibody. In patients with a positive DAT due to to an alloantibody or drug, both the young and old RBC gave an equally reactive DAT, while in patients with a positive DAT due to an alloantibody the young cells were weakly reactive or nonreactive and the older cells were more strongly reactive. When used together, the position of the density distribution curve and the pattern of distribution of antibody coating on young and old RBC provide important diagnostic information about the etiology and clinical status in a patient with a positive DAT and allow for the recognition of an alloantibody and autoantibody when both are present in the same patient.

Recovery of autologous erythrocytes in transfused patients.

A microcapillary method utilizing phthalate esters or an ultracentrifuge method are both capable of separating autologous from homologous erythrocytes in polytransfused patients. The microcapillary technique which is readily adaptable to blood bank laboratories provides a previously unavailable method for defining blood group antigen typings in transfused patients. Such typings are of vital importance in the laboratory evaluation of transfused patients with multiple or weak blood group antibodies.

Sodium and potassium changes in blood bank stored human erythrocytes.

Storage of red cells for three weeks at 4 C under blood bank conditions resulted in a rise in intracellular Na+ and a fall in intracellular K+ with concomitant opposite changes in Na+ and K+ levels in the suspending plasma. A decline in red blood cell ATP during the storage period did not appear to be contributing to the changes. Increasing red blood cell ATP to levels 2 to 3 times normal did not prevent the cation changes from occurring. When assayed at 37 C in the presence of added Mg++, ouabain-sensitive membrane ATPase activity and kinetics of activation by Na+ were unaffected by the three week period of cold storage. However, when assayed at 4 C without added Mg++, simulating the conditions of storage, ATPase activity was negligible. Sodium and potassium did not change when red blood cells with normal ATP content were stored at 20 to 24 C even in the absence of added Mg++. Thus, a major cause for the development of cation changes in the red blood cell during blood bank storage in the temperature which inhibits membrane ATPase, allowing cations to leak unopposed into and out of the red blood cells.

Elevated red blood cell 2,3-diphosphoglycerate levels in black blood donors.

Mean levels of 2,3-diphosphoglycerate (DPG) were significantly increased in erythrocytes (RBC) from 43 nonanemic black blood donors (4.80 +/- 0.06 micromoles/l RBC) compared with 22 white donors 4.47 +/- 0.08 micromoles/l RBCs from eight of the 12 black donors with DPG levels greater than 5 micromoles/l RBC. Although a potentially hemolytic disorder could be defined in four (AS hemoglobin, beta-Thalassemia minor, G6PD deficiency), reticulocyte counts were normal. However, when RBCs from the subgroup were compared to RBCs from an additional 25 unselected white donors, the following suggested an abnormally large population of young RBCs in the subgroup: 1) normal or elevated RBC-ATP with normal serum phosphate level; 2) significantly increased activities of RBC age-dependent enzymes hexokinase (p less than 0.02), pyruvate kinase (p less than 0.05), and glutamicoxaloacetic transaminase (p less than 0.01), with normal activity of phosphoglycerate kinase, an age-independent enzyme; 3) decreased dense (older) RBCs as determined by sedimentation in phthalate esters. Since DPG is increased in young RBCs and falls as the RBC ages, loss of older relatively DPG depleted RBCs due to shortened survival could account for the elevated DPG levels seen in the subgroup.

Effect of alkali-treated lipopolysaccharide on the intracellular cations of human erythrocytes.

The adsorption to human erythrocytes of Escherichia coli lipopolysaccharide treated by mild alkaline hydrolysis (h-LPS) stimulated an increase in the intracellular Na+ concentration and a decrease in the intracellular K+ concentration of the erythrocytes. Erythrocytes treated by h-LPS remained responsive to the membrane adenosine triphosphatase inhibitors ouabain and ethacrynic acid, indicating that hLPS did not alter erythrocyte cations be depleting energy intermediates or uncoupling energy metabolism from active cation transport. The h-LPS-treated erythrocytes became non-agglutinable by the lectin concanavalin A prior to the development of changes in intracellular cations. In addition, h-LPS-treated erythrocytes demonstrated a three-fold greater cation response to ethacrynic acid than the untreated erythrocytes; this greater response was probably due to local membrane effects by h-LPS on the ethacrynic acid-sensitive adenosine triphosphatase. It is suggested that the h-LPS-induced alteration of erythrocyte cation content was secondary to an increase in ion permeability localized to the concanavalin A receptor regions of the erythrocyte membrane, possibly combined with indirect effects of membrane-bound h-LPS on ethacrynic acid-sensitive adenosine triphosphatase.

Hypothermic total body washout and intracranial pressure monitoring in Stage IV Reye syndrome.

The number of children in this report treated with either TBW or exchange transfusions is small. Case mortality rates among children with Reye syndrome in Stage IV coma tends to be exceedingly high, varying from 50 to 100%. Intracranial pressure monitoring with the subarachnoid screw may have been an additional factor in increasing our survival data in three patients in the TBW group, since it provided continuous monitoring of ICP and allowed judicious administration of mannitol intravenously. Survival of five of six patients without neurologic sequelae in the present series has encouraged us to coninue utilization of TBW in children with Stage IV Reye syndrome.

A comparison of a low ionic strength saline medium with routine methods for antibody detection.

Antibody detection studies were undertaken in order to compare a low ionic strength (LIS) medium with a conventional albumin-fortified isotonic medium. Tests were performed in parallel with both media at room temperature and at 37 C. A 30mM NaCl solution was used as the LIS medium and in this study this enhanced antibody reactions without causing nonspecific reactions. The LIS medium detected all of more than 50 Rh and more than 75 non-Rh antibodies after 15 minutes of incubation. Often 30 to 60 minutes of incubation were required to detect these antibodies by the routine method. Several antibodies that were detected with the LIS medium after 15 minutes of incubation were either undetected or had given a nonspecific pattern of activity after 60 minutes incubation in the routine medium. When an antibody was present, the LIS medium invariably gave stronger, more clear-cut results. It is concluded that the LIS medium is generally more sensitive than a conventional medium in detecting antibodies since such a medium will detect clinically significant antibodies after only 15 minutes incubation as well as detect antibodies missed by a conventional medium. An antibody detection system utilizing this medium has obvious applicability to a hospital transfusion service.