Use of a simple, inexpensive device for collection of blood during acute normovolaemic haemodilution in a Jehovah's Witness patient.

Postoperative anaemia is a concern for patients who refuse blood products or have rare blood types. Acute normovolaemic haemodilution (ANH) is a potential solution for these challenging populations. However, protocols for ANH provide limited detail on preparation of blood collection systems. This report describes a novel protocol for ANH and an example of a patient who clearly benefited from ANH.

Algorithms utilizing peripheral blood hematopoietic progenitor cell counts in lieu of some CD34+ cell counts predict successful peripheral blood stem cell collections with substantial time and cost savings.

BACKGROUND AND OBJECTIVES: Hematopoietic progenitor cell (HPC) counts from Sysmex hematology analyzers have been shown to correlate with peripheral blood (PB) CD34+ cell counts by flow cytometry. Algorithms utilizing HPC counts to guide stem cell collections have been proposed but rarely tested. This study describes the development and validation of algorithms utilizing HPC and PB CD34+cell counts to predict adequate peripheral blood stem cell (PBSC) collections for chemomobilized and cytokine-mobilized individuals. MATERIALS AND METHODS: Utilizing a test set of 83 PB samples from chemomobilized or cytokine-mobilized PBSC collection patients, PB CD34+ counts were correlated with HPC counts and a receiver operating characteristic curve was constructed. Cut-offs of ≤0.5 HPC/µl and ≥7 HPC/µl were established to maximize sensitivity and specificity for using HPC to predict PB CD34+ ≥ 10 cells/µl. These cut-offs were subsequently validated using a separate prospective validation set of 88 HPC/CD34+ cell sample pairs. RESULTS: Using the algorithms, all patients in the prospective validation data set achieved adequate collections of ≥1 × 106 CD34+ cells/kg, and a 67% reduction in the number of CD34+ cell counts performed was achieved. This lead to a direct cost savings of at least $18,700 USD over a 21-month period (88% reduction in direct costs). CONCLUSION: Use of the algorithms provides significant time and cost savings for the laboratory while accurately predicting (i) timing of PBSC collections to obtain adequate CD34+ product yields for chemomobilized patients and (ii) when to administer plerixafor to cytokine-mobilized patients to improve the likelihood of achieving adequate collections.

Case report: Eculizumab rescue of severe accelerated antibody-mediated rejection after ABO-incompatible kidney transplant.

ABO-incompatible (ABOI) living donor kidney transplantation has become a well-accepted practice with standard protocols using perioperative antibody-depleting therapies to lower blood group titers to an acceptable threshold for transplantation. However, a subset of patients will experience accelerated antibody-mediated rejection (AMR) after ABOI kidney transplantation and require aggressive intervention to prevent allograft loss. Here in we report the successful use of terminal complement inhibition with eculizumab to rescue an ABOI kidney allograft with accelerated AMR refractory to salvage splenectomy and daily plasmapheresis. This case emphasizes the fact that, despite close postoperative surveillance and aggressive intervention, graft loss from accelerated AMR after ABOI kidney transplantation remains a very real risk. Eculizumab may offer a graft-saving therapeutic option for isolated cases of severe AMR after ABOI kidney transplantation refractory to standard treatment.

Quality activities associated with hospital tissue services.

Quality assurance is a vital component of a tissue service that aims to meet regulatory requirements and provide safe and functional tissue for surgical procedures in the institution. Many hospital transfusion services have or are in the process of assuming tissue service functions for their institutions, and the concepts of tissue quality assurance requirements should be familiar to the transfusion service. This review discusses the key aspects of tissue service quality assurance, including requirements for FDA registration, selection and qualification of tissue suppliers, recordkeeping, management of occurrences and tissue recalls, adverse event reporting, audits, and quality control. Comparing the similarities and differences between tissue and blood component regulatory requirements suggests transfusion service processes can be readily adapted to incorporate quality assurance for tissue service activities.

Logistical aspects of human surgical tissue management in a hospital setting.

Many hospital transfusion services have assumed responsibility for the coordinated management of human allograft tissue. This overview summarizes logistical aspects of tissue management based on the experience of a centralized tissue service at a large academic hospital, in which tissue is stored in a location remote from patient care areas. Operational aspects include determination of which personnel classifications will perform the necessary functions, establishment and maintenance of the standing tissue inventory (including pros and cons of alternative approaches to inventory acquisition), and necessary considerations for making tissue available for surgical cases in the hospital. The nature of communications regarding tissue orders for individual surgical cases is discussed, as well as mechanisms for storage of tissue and transportation and delivery of tissue to the surgical suites. Finally, options for the disposition of tissue that has been dispensed from the tissue service but was not used during the surgical procedure are summarized. With attention to these details, a tissue service can provide reliable, high-quality tissue in a timely fashion.

Recurrent transfusion-related acute lung injury after a two-year interval.

Transfusion-related acute lung injury (TRALI) is a life-threatening complication of blood transfusion. The epidemiology and pathogenesis of TRALI are not well established. A Medline literature search shows only rare reports of recurrent TRALI, all occurring soon after the first episodes. We report a case of recurrent TRALI after a 2-year interval. A patient developed TRALI after transfusion of 4 units of fresh frozen plasma for gastrointestinal bleeding due to oesophageal varices in September 2002. The patient required mechanical ventilation but recovered completely. Two years later, in October 2004, the patient experienced a second episode of TRALI during liver transplantation for hepatitis C virus /alcoholic cirrhosis. Again, the patient recovered after ventilator support. Laboratory investigation of the first TRALI episode (2002) showed antibodies against class II human leukocyte antigens (HLA) in three female donors. Laboratory investigation of the second episode (2004) showed anti-DR52 (HLA class II) antibodies in one female donor matching the DR-52 HLA class II antigen in the recipient. TRALI can rarely recur. Consideration of future blood needs for patients experiencing recurrent TRALI should include preventive measures against further TRALI reactions, such as blood from male donors or blood less than 14 days old.

Type I interferon is the primary regulator of inducible Ly-6C expression on T cells.

Ly-6C has been proposed as a marker of memory CD8+ T cells. Reports have indicated that Ly-6C is upregulated after T cell receptor (TCR) stimulation or exposure to proinflammatory cytokines. This study examined the relative roles of proinflammatory cytokines and TCR engagement in Ly-6C induction. In vitro experiments tested the effects of cytokines on Ly-6C expression and confirmed interferon-alpha (IFN-alpha) as a primary cytokine that induces Ly-6C expression on CD4+ and CD8+ T cells. The amount and duration of Ly-6C expression were examined on T cells after in vivo induction of proinflammatory cytokines (CpG oligodeoxynucleotides [ODN]) or TCR activation (staphylococcal enterotoxin B [SEB]). In vivo, proinflammatory cytokines transiently upregulated Ly-6C on T cells in the absence of TCR stimulation. TCR stimulation by SEB transiently upregulated Ly-6C expression on antigen-specific and antigen-nonspecific T cells but did not cause long-term upregulation of Ly-6C expression in either population. IFN-alpha was confirmed as a primary inducer of Ly-6C in vivo, as CpG ODN were unable to induce Ly-6C expression in IFN-alphaRI(-/-) mice. Thus, inducible Ly-6C expression on CD4+ and CD8+ T cells is largely due to IFN-alpha in the environment and appears not to be directly correlated with the development of T cell memory.

Delineation among eight major hematopoietic subsets in murine bone marrow using a two-color flow cytometric technique.

BACKGROUND: Many methods have been developed specifically for identifying hematopoietic progenitor cells in murine bone marrow, but few methods allow rapid identification of multiple bone marrow populations. We describe a new, simple method for identifying simultaneously eight populations in murine bone marrow with two-color flow cytometry and phenotypically define these populations. METHODS: Bone marrow was stained with anti-Ly-6C and anti-B220 (CD45R) in one fluorochrome and wheat germ agglutinin (WGA) in another fluorochrome. The eight populations identified in this way were defined further primarily by four-color flow cytometry. RESULTS: Six of the eight populations were characterized phenotypically as containing erythroid, granulocytic, mast, early B, mature B, and stem cell populations. Two additional populations with phenotypic characteristics of partially differentiated precursor cells also were identified. One population was Ly-6C/B220+ and WGA-. It also expressed markers associated with early B, T, and/or dendritic cell differentiation. The second population was Ly-6C(hi)WGA(hi)Mac-1+ and was negative for numerous other lineage-specific and precursor markers. Its morphology suggested monocytic differentiative potential. CONCLUSIONS: A two-color flow cytometric assay profiles six bone marrow populations with identifiable phenotypes and two additional unique, putative hematopoietic precursor populations.

Outcome of transfusion of K:11 erythrocytes in a patient with anti-K11 antibody.

BACKGROUND AND OBJECTIVES: The clinical significance of anti-K11 in red cell transfusion therapy is unknown. We report the outcome of transfusion of K:11 erythrocytes into a patient with a known anti-K11 antibody. MATERIALS AND METHODS: The patient was monitored clinically following transfusion of 11 units of K:11 erythrocytes. A red cell survival study with K:11 erythrocytes and a monocyte monolayer assay (MMA) were performed. RESULTS: No adverse clinical outcome was detected. The red cell survival study showed normal survival of K:11 erythrocytes, and the MMA showed no increase in reactive monocytes. CONCLUSION: These findings suggest that K:11 red cells can safely be transfused to individuals with anti-K11 antibody.

Distribution of Ly-6C on lymphocyte subsets: I. Influence of allotype on T lymphocyte expression.

The expression patterns of the Ly-6C Ag were examined on splenic and thymic lymphocyte subsets of Ly-6.1 and Ly-6.2 strains of mice using the rat mAb 15.1. Ly-6C is expressed on subsets of CD4+ and CD8+ splenocytes, and a portion of NK cells. Within the splenic and lymph node CD4+ T cell compartment, Ly-6C expression is restricted to Ly-6.2 strains of mice, and is present on a subset of naive cells. Ly-6C is expressed on the majority of peripheral CD8+ T cells in both Ly-6.1 and Ly-6.2 strains, and is found primarily on the Ag-experienced subset. In the thymus, Ly-6C is present on subpopulations of CD4- CD8+, CD4- CD8-, and CD4+ CD8- cells. Ly-6C+ CD4- CD8+ thymocytes show a mature phenotype, while Ly-6C+ CD4- CD8- and Ly-6C+ CD4+ CD8- thymocytes appear to be part of the recently described NK1.1+ alphabeta TCR+ population. On account of the marked differences in Ly-6C expression on peripheral CD4+ T cells from Ly-6.1 and Ly-6.2 strains of mice, additional experiments were undertaken to assess Ly-6C expression in parental and Ly-6.1 x Ly-6.2 F1 mice. Neither phenotype dominated in the F1 offspring, with frequencies of Ly-6C+ CD4+ splenocytes falling in the intermediate range. Further experiments compared the staining patterns of the rat anti-pan Ly-6C (Ly-6.1 and Ly-6.2) Ab with a mouse anti-Ly-6.2 allotype specific Ab, with emphasis on both Ly-6.2 and Ly-6.1 x Ly-6.2 F1 mice. The results demonstrate the presence of lymphocytes that express the pan form of Ly-6C but not the form recognized by the alloantibody. This latter finding suggests the presence of more than one form of the Ly-6C Ag.

Evaluation of a new protamine titration method to assay heparin in whole blood and plasma.

When unfractionated heparin is used for therapeutic anticoagulation, the heparin effect must be monitored to avoid thrombotic or hemorrhagic complications. The ability of a factor Xa inhibition (XaI) assay was compared with that of a low-level heparin protamine titration (LLHPT) assay to measure the concentration of heparin after heparin was added in vitro to specimens of plasma and whole blood. Heparin effect on the activated partial thromboplastin time also was assessed in the same specimens. The XaI and LLHPT assays had comparable precision and provided linear results over a wide range of heparin concentrations. Both assays slightly underestimated the total amount of heparin added to the specimens. The most rapid test was the whole blood LLHPT assay; this test therefore may be useful for bedside monitoring of heparin. A significant disadvantage of the LLHPT assay was the large sample size required to perform it. These results provide in vitro evidence that the XaI and LLHPT assays can provide equally precise monitoring of heparin concentration.

Behavior of the idiotypic network in conventional immune responses. I. Kinetics of idiotypic and anti-idiotypic antibodies following immunization with T-independent and T-dependent antigens.

A minimal requirement in investigations of the behavior of the idiotypic network during immunization is the ability to quantitate both the idiotypic (Ab1) and anti-idiotypic (Ab2) responses. Quantitation of Ab2 in serum is complicated by the simultaneous presence of Ab1, so that Ab1-Ab2 immune complexes escape detection. In contrast, immune complexes should not complicate the enumeration of Ab2-producing lymphocytes in a hemolytic plaque assay. This study utilizes a procedure that allows detection of Ab2-producing cells in such an assay. The procedure relies upon the insertion of the appropriate antibody (Ab1) into the membrane of indicator SRBC through a covalently attached dipalmitoyl phosphatidylethanolamine (DPPE) tail. When the Ab2 response following murine immunization with DNP-Ficoll was analyzed using such an assay, peak plaque-forming cell (PFC) numbers were found to coincide with peak Ab1 PFC numbers in both the primary and secondary response. In addition, this Ab2 response was found to be T independent. The murine immune response to DNP-HGG demonstrated a peak Ab2 PFC response which followed the peak Ab1 PFC response after both primary and secondary immunization. This Ab2 response appeared to be T dependent. The secondary responses to both DNP-Ficoll and DNP-HGG showed increased levels of Ab2 PFC and decreased levels of Ab1 PFC in comparison to the primary responses to the same antigens, suggesting that immunoregulation may occur within these idiotypic networks.

Behavior of the idiotypic network in conventional immune responses. II. Affinity and heterogeneity of idiotypic and anti-idiotypic antibodies following immunization with T-independent and T-dependent antigens.

The relative affinity and heterogeneity of affinity of idiotypic and anti-idiotypic antibodies in mice immunized with the T-independent antigen DNP-Ficoll and the T-dependent antigen DNP-HGG were measured by a plaque inhibition assay. Idiotypic plaque-forming cells (PFC) were detected by a conventional assay utilizing DNP-coated SRBC. Anti-idiotypic PFC were detected with SRBC coated with affinity-purified anti-DNP antibody of rabbit origin. It was found that both idiotypic and anti-idiotypic antibodies elicited by immunization with the T-independent antigen had lower affinity and were less heterogeneous than the corresponding antibodies originating in mice immunized with the T-dependent antigen. In addition, the affinity and heterogeneity values of the idiotypic antibodies were correlated with the affinity and heterogeneity values of the anti-idiotypic antibodies from the same mice. This finding indicates that idiotypic and anti-idiotypic antibodies mutually regulate each other, thus pointing to internal immunoregulatory effects of the idiotypic network with respect to these parameters.

Detection and enumeration of immunoglobulin secreting cells.

A method is described in which sheep red blood cells (SRBC) are coated with anti-immunoglobulin (Ig) antibodies (Ab) for use in reverse hemolytic plaque assays (RHPA) as follows. The non-complement fixing F(ab')2 fragments of rabbit anti-mouse Ig Ab are derivatized with the N-hydroxysuccinimide ester of palmitate. The hydrophobic palmitate tails spontaneously insert into the SRBC membranes, thus coating the cells with anti-Ig F(ab')2 molecules. The SRBC are lysed by successive additions of mouse Ig, rabbit anti-mouse Ig and complement. When this procedure is carried out in agar gel, Ig-secreting mouse cells produce localized hemolytic areas (plaques). The procedure is more reproducible and more sensitive than RHPA performed with protein A-coated SRBC. In principle, this procedure should be adaptable to the detection of cells secreting any molecule for which specific antibodies are available.