RBC Components Research Articles

Platelet alloimmunization after long‐term red cell transfusion in transfusion‐dependent thalassemia patients

The platelet (PLT) alloimminization status after long-term red cell (RBC) transfusion in thalassemia patients was investigated, including antibodies against HLA antigens and PLT-specific glycoprotein antigens. Blood samples from a total of 60 thalassemia patients who routinely received washed RBCs were tested for the presence of HLA antibodies and PLT-specific glycoprotein antibodies with a commercial enzyme immunoassay kit. All patients were rescreened at a follow-up period of 12 to 15 months. At the first year of study, 19 (31%) patients had HLA antibodies, 13 (22%) had HLA antibodies and PLT-specific antibodies, and 1 (2%) had PLT-specific antibodies. One patient showed weak reactive PLT autoantibody. The follow-up study showed that 7 patients developed HLA antibodies, whereas 1 patient lost HLA antibody activity. Nine patients developed new PLT-specific antibodies, yet 12 patients lost at least one of their PLT-specific antibodies. Long-term RBC transfusions can induce PLT alloimmunization, both to HLA antigens and to PLT-specific antigens. The residual PLTs and white blood cells in RBC components could be the sources of immunization. In our thalassemia patients, HLA antibodies likely sustain longer than PLT-specific antibodies.

強力化学療法と同種骨髄移植療法が施行し得たｌｇＡ欠損症を伴う急性骨髄性白血病の１症例

Deficiency of IgA with circulating class-specific anti-IgA antibodies is one of the major causes of anaphylactic reaction in transfusion medicine. This problem is more serious in the management of patients with hematological malignancies or other types of bone marrow failure. Here we report a case of acute myeloid leukemia with IgA deficiency who underwent intensive chemotherapy and allogeneic bone marrow transplantation. To avoid the production of anti-IgA antibody, platelet concentrates were collected from donors who were IgA-deficient. Additional support with platelets and RBC components from donors who had circulating IgA was provided using products washed and replaced with 0.9% normal saline. Complete remission was achieved after a single course of remission induction therapy. After one course of consolidation chemotherapy, allogeneic bone marrow transplantation from an HLA-matched sibling was performed. No anaphylactic reaction was observed after any transfusion. No circulating anti-IgA antibodies were detectable after the full recovery of hematopoiesis in this patient. These findings indicate that allogeneic blood component products collected from donors with IgA deficiency are useful in myeloablative therapy in patients with hematological disorders. By washing with normal saline, products from donors with normal IgA levels are also acceptable. Further studies to elucidate the clinical usefulness of precise genomic and expressional analysis of IgA gene are required to ensure the safety of transfusion medicine in patients with IgA deficiency. Furthermore, the establishment of blood bank registry systems for IgA-deficient blood donors is important to the maintenance of a stable supply of blood components for IgA-deficient patients who require transfusion therapy.

Effects of rejuvenation and frozen storage on 42-day-old AS-3 RBCs.

A storage period of 42 days has been approved by the FDA for RBCs stored in NUTRICEL (AS-3). This study was undertaken to provide data to the FDA about the feasibility of salvaging AS-3 RBCs at the end of their storage period by rejuvenation and freezing, and to evaluate the effect of rejuvenation on indicators of RBC function. Healthy adults (n = 22) donated 450 mL of whole blood, and RBC components were prepared at two study sites. The components were stored at 1 to 6 degrees C for either 41 days (nonrejuvenated frozen controls, n = 6) or 42 days (rejuvenated frozen study group, n = 10; nonrejuvenated nonfrozen controls, n = 6). Rejuvenated study components and nonrejuvenated frozen controls were stored at -70 degrees C for longer than 2 weeks. Frozen units were then deglycerolized and kept for an additional 24 hours at 1 to 6 degrees C. 2,3-DPG, and ATP were reduced after 42-day storage to near 0 and 65 percent, respectively, of their original values. After rejuvenation and deglycerolization, the mean ATP level was 146 percent and the mean 2,3-DPG was 115 percent. The percent freeze-thaw-wash recovery was similar for rejuvenated and nonrejuvenated RBCs. Trace amounts of hypoxanthine and inosine were detected in rejuvenated units. The mean 24-hour survival (single- or double-label technique) of all components exceeded 75 percent. The t1/2 of study and control RBCs was similar. The ability of 42-day-old AS-3 RBCs to deliver oxygen after rejuvenation and freezing is not impaired. These data indicate that rejuvenated AS-3 RBCs can provide a safe and beneficial blood component immediately upon infusion.

Increasing oxygen tension improves filtration of sickle trait donor blood.

A major cause of filter failure of red cell (RBC) components from donors with sickle cell trait (HbAS) is the polymerization of haemoglobin. The oxygen saturation (sO2) of blood stored in various plastics and different volumes of air was assessed. Blood from 10 HbAS donors was collected and divided into two bags, one with air added, one without. Bags with added air had increased sO2 levels (from 49 +/- 10% to 76 +/- 6%). Filtration was successful for nine of 10 components with air, and one of 10 without air. Successful filtration of RBC components occurs when sO2 is increased.

WBC-containing allogeneic blood transfusion and mortality: a meta-analysis of randomized controlled trials.

An association between allogeneic blood transfusion (ABT) and mortality was reported by one team of investigators from randomized controlled trials (RCTs) comparing recipients of non-WBC-reduced versus WBC-reduced RBCs in open-heart surgery. A meta-analysis was undertaken to examine whether this finding can be generalized across clinical settings and/or transfused RBC components. RCTs reporting on adverse immunomodulatory effects of ABT between January 1992 and August 2002 were retrieved. Fourteen studies had recorded mortality as a primary or secondary outcome and met all other criteria for meta-analysis. Summary ORs of mortality in a treatment arm receiving WBC-containing allogeneic RBCs versus a control arm receiving autologous or WBC-reduced allogeneic RBCs were calculated across studies, for groups of studies in which the hypothesis of homogeneity was not rejected. There was no association between ABT and mortality across 14 RCTs reporting on short-term mortality (summary OR, 1.20; 95% CI, 0.87-1.65) or across 3 RCTs reporting on long-term mortality (summary OR, 0.87; 95% CI, 0.64-1.19). In subgroup analyses, RCTs using autologous blood or conducted in abdominal or vascular surgery showed no difference in mortality, but short-term mortality differed across 3 RCTs conducted in open-heart surgery (summary OR, 2.26; 95% CI, 1.31-3.90; p < 0.05) and 7 RCTs comparing recipients of non-WBC-reduced versus WBC-reduced allogeneic RBCs filtered before storage (summary OR, 1.45; 95% CI, 1.00-2.11; p >/= 0.05). An association between ABT and either short-term or long-term mortality was not detected across clinical settings and transfused RBC components, but subgroup analyses suggest that an association between WBC-containing ABT and short-term mortality may exist in open-heart surgery and in settings where WBC-reduced allogeneic RBCs filtered before storage are administered.

Frozen preoperative autologous blood donation for heart transplantation at the Mayo Clinic from 1988 to 1999.

Preoperative autologous blood donation (PABD) has been used to reduce the need for allogeneic RBC transfusion, decreasing risk and conserving supply. A frozen PABD program for heart transplant patients was instituted at the Mayo Clinic in 1988, but participation has steadily declined. The aims of this study were to determine how the availability of PABD influenced the transfusion RBC components, whether the availability of PABD reduced exposure to allogeneic RBC components, and the costs of providing PABD units. A retrospective review of all heart transplant cases from 1988 to 1999 was performed (n = 141). Data on collection and transfusion practices were compared for patients with (n = 88, 62%) and without PABD (n = 53, 38%). Total RBC transfusion requirements did not differ between the groups. Patients with frozen PABD received fewer allogeneic units, but they also had less blood salvaged and reinfused. Twenty patients (23%) completely avoided exposure to allogeneic RBCs in the PABD group versus three patients (6%) in the group without PABD. Although patients in the PABD group successfully donated a total of 423 units, 41 percent were discarded. Over 11 years, the need for 251 units of allogeneic RBCs was avoided ($27,610), but $283,500 was spent to have the frozen PABD units available. PABD can be performed for heart transplantation, but it is expensive.

Sickle Hb polymerization in RBC components from donors with sickle cell trait prevents effective WBC reduction by filtration.

RBC components collected from donors with sickle cell trait frequently occlude WBC-reduction filters. In vitro, sickle trait RBCs have the potential for sickle Hb (Hb S) polymerization at low oxygen saturations and high Hb concentrations. To determine if the low pH and high osmolarity of the CP2D used in the collection contributed to filter failures, the filterability of sickle trait donor RBCs collected in CP2D was compared with RBCs from the same donors collected in heparin. Five of six sickle trait components collected in CP2D did not complete filtration, but all six RBC components collected in heparin filtered completely. RBC components collected in CP2D from four other sickle trait donors were divided in two, and one-half was treated with carbon monoxide to convert Hb S to its liganded form to prevent Hb S polymerization. All four carbon monoxide-treated components filtered within 9 minutes, but only one untreated component filtered completely. RBC components collected by apheresis contained less CP2D, and five of seven sickle trait apheresis components filtered completely; four of the five filtered rapidly (<15 min) and one filtered in 100 minutes. Hb oxygen saturation was greater in the four rapidly filtering apheresis RBC components (68 +/- 9%) than in the three that filtered slowly or incompletely (37 +/- 5%, p = 0.03). Hb S polymerization appears responsible for RBC WBC-reduction filter failures. Citrate anticoagulant and low oxygen saturation are responsible in part for Hb S polymerization in this setting.

Monitoring for underutilization of RBC components and platelets.

Monitoring blood transfusion for overutilization is standard practice at most institutions. This study monitored for underutilization of blood transfusion over a 14-month period, by evaluating patients who had Hb levels that were reported to be <5 g per dL or platelet counts <10 x 10(9) per L and who did not receive an RBC or platelet transfusion within 24 hours of the reported results. During the study period, 24,004 units of RBCs and 3,967 units of apheresis platelets were transfused. There were 148 patients who had a Hb level that was reported to be <5 g per dL or a platelet count reported to be <10 x 10(9) per L and who did not receive a transfusion during the 24 hours after the reporting of these results. In 5 cases, the patients died before the reporting of the low Hb or platelet counts, which precluded the low Hb or low platelet count reports from triggering transfusion therapy. In 8 cases, an underutilization review investigation could not be done, because of the unavailability of patient charts. Of the remaining 135 cases, investigation revealed justifiable reasons for withholding transfusion in 133. In 2 cases, the withholding of transfusion was deemed by peer review to be inappropriate, as the patients should have received a transfusion. Overall, there was one documented underutilization of RBC transfusion therapy during a period when 24,004 units were transfused and one underutilization of platelet transfusion therapy during a period when 3,967 units of apheresis platelets were transfused. Monitoring for underutilization of transfusion therapy fulfills the requirements of the Joint Commission on the Accreditation of Healthcare Organizations: While the underutilization of transfusion therapy did not appear to be a significant problem at this medical center, determining the reasons for withholding transfusions shed light on important patient care-related issues, including preexisting causes of falsely low platelet counts and Hb levels, delays in investigating critical laboratory values, and the need for policies for the treatment of patients who refuse transfusion for personal or religious reasons.

Alloimmunization in preterm infants after repeated transfusions of WBC-reduced RBCs from the same donor.

Preterm infants are among the most heavily transfused of patient groups, yet multiply transfused infants only rarely produce alloantibodies against RBC or WBC antigens. It is not known whether rates of alloimmunization might be increased by repeated exposure to RBCs and WBCs from the same donor, as in limited-donor-exposure programs, or whether infants might benefit from WBC-reduced RBC components as a means of diminishing the risk of possible alloimmunization. Preterm infants (birth weight 0.6-1.3 kg) received prestorage WBC-reduced RBCs from dedicated donors, collected in AS-3 as a means of limiting donor exposures. Blood samples were collected serially from infants shortly after birth until either discharge or age 6 months and were studied for RBC and WBC antibodies-the latter with reactivity against either HLA class I or neutrophil-specific antigens. Thirty preterm infants received 139 transfusions (mean, 4.6; median, 4 transfusions per infant), with 81 percent of transfusions obtained from one donor per infant. Eighty-four blood samples (mean, 2.7/infant) were studied, and no infant produced RBC antibodies. Twenty-seven percent of infants exhibited WBC antibodies, but only 13 percent actually produced WBC antibodies (passive maternal antibody excluded). Of the WBC antibodies produced by infants, three were against HLA class I and one was against neutrophil-specific antigens; none were linked to adverse effects. Because infants only rarely produce RBC antibodies, no changes in blood banking practices are necessary for limited-donor-exposure programs. Although the production of WBC antibodies by infants occurs, it seems to be uncommon; thus, the possible benefits, if any, of WBC reduction are uncertain, and further study is required before changes in practice can be justified.

Cord blood as a source of autologous RBCs for transfusion to preterm infants.

This prospective study was conducted to gain experience as to whether it is technically possible to produce autologous RBCs in additive solution from cord blood (CB), to optimize the blood supply for preterm infants. CB was collected from 47 infants with a mean (+/- SD) birth weight of 1717 (+/- 699) g. Whenever possible, RBC components were prepared by standard centrifugation using a six-bag system. All samples were put in sterility testing quarantine for 5 days, and a maximum storage of 14 days from collection to transfusion was specified. The babies were given either the autologous RBCs or standard allogeneic RBC concentrates, if autologous blood was not available. In 81 percent of the samples, autologous RBC components could be processed (vol, 7-87 mL; Hct, 31-82%). But within the group of extremely low birth weight infants (body weight <1000 g), a mean CB net volume of only 37 mL was collected, and the RBC preparation was successful only in exceptional cases. Three CB samples (8.6%) tested positive in sterility testing. Of the 47 infants, 21 were treated with a total of 62 allogeneic and 4 autologous RBC transfusions. Most infants with a body weight over 1400 g did not need any RBC transfusion. The preparation of autologous RBCs from the CB of preterm infants is technically possible in principle. However, major concerns must be raised as to whether such preparations are of benefit in ensuring safe care of neonates with blood components, with respect to the high rate of bacterial contamination and the limited availability in babies with low birth weight.

Effects of methylene blue-treated plasma on red cells and stored platelet concentrates.

Photochemical methods can effectively inactivate extracellular viruses and bacteria found in blood components. Treatment of plasma with methylene blue (MB), a phenothiazine dye, and visible light inactivates enveloped viruses including HIV-1. The effects of MB-treated plasma on cellular components stored in vitro have not been well characterized. MB-treated plasma (83 microg MB/250 mL plasma) was added to single-donor platelets, stored AS-1 red cells (RBCs), irradiated RBCs, and frozen-deglycerolized RBCs. In vitro platelet assays performed after 1 and 5 days of storage in MB-treated plasma included pH, pO2, pCO2, HCO3, platelet number, lactate dehydrogenase, glucose, osmotic recovery, and CD62 expression. RBC components were examined at specific intervals for leakage of potassium, plasma hemoglobin level, and percentage of hemolysis. Direct antiglobulin tests, osmotic fragilities, and RBC antigen stability tests were also performed on RBCs stored in MB-treated plasma. Components stored with autologous plasma or nontreated allogeneic plasma served as controls. Similar storage-induced changes in pH, glucose, and platelet numbers, as well as increases in lactate dehydrogenase, CD62 expression, and lactate were seen in single-donor platelets stored with MB-treated and control plasma. Platelet morphology scores and osmotic recoveries were not altered. Plasma hemoglobin and potassium and percentage of hemolysis increased equally in the various RBC components stored with MB-treated or nontreated plasma. Osmotic fragility and RBC antigen stability were not appreciably altered by MB-treated plasma. Plasma treated by MB photoinactivation can be used for in vitro resuspension and storage of platelets or RBCs, because of the lack of influence of MB-treated plasma on a variety of in vitro platelet and RBC assays.

Marked increase in beta-tubulin mRNA expression during regeneration of axotomized retinal ganglion cells in adult mammals

Changes in gene expression were investigated in axotomized CNS neurons under conditions that inhibit or permit regrowth of their damaged axons. Levels of mRNA encoding beta-tubulin, the 150 kDa neurofilament subunit (NF-M), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were examined by quantitative in situ hybridization of adult rat retinal ganglion cells (RGCs) after axotomy in the optic nerve or during regeneration in a peripheral nerve (PN) graft. Soon after optic nerve section beta-tubulin, NF-M, and GAPDH mRNA levels decreased and remained low during the 1 month studied. In these retinas beta-tubulin mRNA fell to approximately 50% of normal controls. However, in the PN-grafted retinas, where approximately 20% of the surviving axotomized RGCs regenerate their axons, there were "hot spots" of beta-tubulin mRNAs where neuronal levels were nearly 300% higher than in controls. By retrograde neuronal labeling these hot spots were shown to correspond to the injured RGCs that regrew their axons into the PN graft; beta-tubulin mRNA levels in nonregenerating RGCs of the same retinas averaged 63% of controls. We suggest that interactions of RBC axons and components of the grafts' non-neuronal environment play a key role in the over fourfold differences in beta-tubulin mRNA levels observed between injured and regenerating RGCs.

Autologous IgM, IgA, and Complement Binding to Sickle Erythrocytes In Vivo. Evidence for the Existence of Dense Sickle Cell Subsets

We have previously reported that sickle erythrocytes sedimenting at high specific density after gradient centrifugation exhibit increased IgG binding in vivo as compared with low-density paired samples. We have performed the present study to determine whether the opsonization of dense sickle cells in vivo could also involve autologous IgM, IgA, and complement. IgA, IgM, and complement binding in vivo to the surface of density-separated sickle erythrocytes was detected by flow cytometric analyses. IgM and complement C3 fragment binding was detected primarily on high-density sickle erythrocytes. With the exception outlined below, IgA binding was detected for all sickle cell fractions that sediment at densities > 1.085 g/mL. IgM, IgA, and complement C3 fragment binding was increased on high-density sickle erythrocytes as compared with low-density paired samples and exceeded that binding to normal erythrocytes by 30% +/- 10% (mean +/- range), 50% +/- 10%, and 41% +/- 5%, respectively. Two-color flow cytometry indicates that high-density sickle cell fractions contain at least two heterogeneous RBC subsets. One is an RBC subset that binds IgA in combination with IgM and C3, and the second subset is devoid of IgA yet binds IgM and C3. These findings indicate that high-density sickle cells exhibit a greater heterogeneity than has been reported in previous studies, which is based on autologous Ig binding in vivo; and suggest that RBC components of this most severely dehydrated sickle cell subpopulation could have heterogeneous origin and pathophysiologic significance. Although the functional role of IgA binding to human RBCs is unclear, our findings that IgM and complement bind to the same high-density sickle cell fractions suggest that both the IgM and the sickle erythrocyte-bound IgG determined in previous studies could mediate the deposition of complement on dense sickle cells in vivo. These findings support the hypotheses that irreversibly sickled cell-enriched high-density sickle RBC subpopulations could be removed from the circulation by erythrocyte phagocytosis that is enhanced by the presence of complement.

Autologous IgM, IgA, and complement binding to sickle erythrocytes in vivo. Evidence for the existence of dense sickle cell subsets

We have previously reported that sickle erythrocytes sedimenting at high specific density after gradient centrifugation exhibit increased IgG binding in vivo as compared with low-density paired samples. We have performed the present study to determine whether the opsonization of dense sickle cells in vivo could also involve autologous IgM, IgA, and complement. IgA, IgM, and complement binding in vivo to the surface of density-separated sickle erythrocytes was detected by flow cytometric analyses. IgM and complement C3 fragment binding was detected primarily on high-density sickle erythrocytes. With the exception outlined below, IgA binding was detected for all sickle cell fractions that sediment at densities &gt; 1.085 g/mL. IgM, IgA, and complement C3 fragment binding was increased on high-density sickle erythrocytes as compared with low-density paired samples and exceeded that binding to normal erythrocytes by 30% +/- 10% (mean +/- range), 50% +/- 10%, and 41% +/- 5%, respectively. Two-color flow cytometry indicates that high-density sickle cell fractions contain at least two heterogeneous RBC subsets. One is an RBC subset that binds IgA in combination with IgM and C3, and the second subset is devoid of IgA yet binds IgM and C3. These findings indicate that high-density sickle cells exhibit a greater heterogeneity than has been reported in previous studies, which is based on autologous Ig binding in vivo; and suggest that RBC components of this most severely dehydrated sickle cell subpopulation could have heterogeneous origin and pathophysiologic significance. Although the functional role of IgA binding to human RBCs is unclear, our findings that IgM and complement bind to the same high- density sickle cell fractions suggest that both the IgM and the sickle erythrocyte-bound IgG determined in previous studies could mediate the deposition of complement on dense sickle cells in vivo. These findings support the hypotheses that irreversibly sickled cell-enriched high- density sickle RBC subpopulations could be removed from the circulation by erythrocyte phagocytosis that is enhanced by the presence of complement.

Studies on the induction of anterior chamber-associated immune deviation (ACAID). 1. Evidence that an antigen-specific, ACAID-inducing, cell-associated signal exists in the peripheral blood.

The anterior chamber of the eye is an immunologically privileged site. Recent evidence indicates that this privilege is an actively acquired immune state in which a unique form of systemic immune deviation exists, anterior chamber-associated immune deviation (ACAID). ACAID is characterized in part by the generation of Ag-specific splenic T lymphocytes that mediate suppression of induction and expression of delayed hypersensitivity and that suppress production of C-fixing antibodies. Delayed hypersensitivity and C fixation are usually associated with extensive nonspecific inflammation and innocent bystander tissue injury. It is, therefore, believed that ACAID represents physiologic adaptations of the immune system that mitigate wanton destruction of the anatomically delicate visual axis, thereby preserving sight, while at the same time providing selective immune protection. As a means of examining the potential contribution of the eye to ACAID induction, we have studied the immune properties of blood harvested from mice that had received BSA into the anterior chamber 48 h earlier. We found that an Ag-specific "signal" is present in the blood of these mice; when blood was transfused into naive syngeneic mice, regulatory cell populations were induced and the recipients were unable to display BSA-specific delayed hypersensitivity. Further analysis of this signal revealed it to be associated with the leukocyte fraction, but not with either the plasma or RBC components of whole blood. Among leukocytes, the suppression-inducing activity correlated positively with cells bearing the mature macrophage marker F4/80 and negatively with cells bearing Thy-1, surface Ig, and class II MHC molecules. These findings are discussed with regard to the potential intraocular sources of the ACAID-inducing signal and the possible mode of action of this factor.

Inhibition of cell-free oxidative bactericidal activity by erythrocytes and hemoglobin.

Sickle cell anemia and other chronic hemolytic anemias are associated with an increased frequency of bacterial infections. There is evidence to suggest that in hemolytic states massive erythrocyte (RBC) ingestion by macrophages interferes with their antibacterial function, thereby predisposing infection. Stimulated by this possibility, we recently demonstrated that erythrophagocytosis by macrophages markedly inhibited intracellular killing of bacteria, and that zymosan-stimulated superoxide generation and chemiluminescence were also suppressed by RBC ingestion. We examined the effects of RBC components on generation of chemiluminescence, superoxide, and bactericidal activity by cell-free oxidative systems. Generation of chemiluminescence by hypoxanthine-xanthine oxidase was depressed in the presence of human RBC lysate or column-fractionated hemoglobin but not crystallized human hemoglobin (methemoglobin) (peak cpms of 15,522 [P = 0.00024], 28,360 [P = 0.0088], and 50,041 [P = 0.37], respectively, compared with 59,898 for positive controls). Similarly, hypoxanthine-xanthine oxidase production of superoxide was inhibited in the presence of column-fractionated human hemoglobin (43.8 versus 17.4 nmol per tube, P = 0.000001). A cell-free bactericidal system, acetaldehyde and xanthine oxidase with or without myeloperoxidase and Cl-, was markedly inhibited by column-purified hemoglobin. For example, after 2 h of incubation, surviving numbers of Staphylococcus aureus were: control (buffer only), 2.5 X 10(6)/ml; bactericidal system, none; bactericidal system plus hemoglobin, 2.2 X 10(6)/ml (P less than or equal to 0.03, bactericidal system versus other systems). Our studies have documented that interactions between RBC (hemoglobin) and reactive products of oxygen metabolism inhibit oxidative bactericidal mechanisms in cell-free systems as well as in macrophages.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation of Plasmodium berghei by Hemolysin Lysis of Infected Erythrocytes and Evidence for a Parasite Hexokinase

A rapid and simple procedure has been developed for the isolation of Plasmodium berghei parasites from infected-mouse erythrocytes employing the heat stable hemolysin produced by Pseudomonas aeruginosa. Using parasites isolated by this method, the presence of a parasite specific hexokinase has been demonstrated, providing an explanation for the increased glucose consumption observed with infected cells. Enzyme assays and serology were employed in determining the purity and yield of purified parasites. The enzyme assays showed that about 25% of the parasites in infected RBCs were recovered in the purified state. The purified parasites were not agglutinated by rabbit-anti-mouse RBC serum which indicated the purified parasites were not contaminated by RBC components.

Distribution of virus in blood components during the viremia of measles.

Measles virus was isolated from four cases of human measles 8 hours to 29 hours after the onset of exanthem when the washed leucocyte fraction of blood was employed as inoculum, but isolation was not obtained from the RBC or plasma components even with larger inocula. In one case, virus was isolated from all fractions of blood drawn at the onset of rash, but in significantly higher quantity from the WBC component.