An introduction to chemokines and their roles in transfusion medicine.

Chemokines are a set of structurally related peptides that were first characterized as chemoattractants and have subsequently been shown to have many functions in homeostasis and pathophysiology. Diversity and redundancy of chemokine function is imparted by both selectivity and overlap in the specificity of chemokine receptors for their ligands. Chemokines have roles impacting transfusion medicine in haematopoiesis, haematologic malignancies, transfusion reactions, graft-versus-host disease, and viral infections. In haematopoietic cell transplantation, chemokines are active in mobilization and homing of progenitor cells, as well as mediating T-cell recruitment in graft-versus-host disease. Platelets are rich source of chemokines that recruit and activate leucocytes during thrombosis. Important transfusion-transmissible viruses such as cytomegalovirus and human immunodeficiency virus exploit chemokine receptors to evade host immunity. Chemokines may also have roles in the pathophysiology of haemolytic and non-haemolytic transfusion reactions.

On a much higher than reported incidence of anti-c in R1R1 patients with anti-E.

A previous study involving tube IATs, untreated RBCs, and a low ionic-strength additive reagent revealed that approximately one-third of R(1)R(1) patients with anti-E have a concomitant anti-c. However, the current study finds a much higher incidence of anti-c in such patients, using gel technology in conjunction with ficin-pretreated RBCs. Results of antibody identification studies and transfusion records of 82 R(1)R(1) patients with anti-E were reviewed. Serologic test methods included a LISS wash solution for tube IATs (15 min at 37 degrees C, anti-IgG), ficin-tube IATs (30 min at 37 degrees C, anti-IgG + anti-C3), and gel IATs (untreated or ficin-treated RBCs or both, anti-IgG gels). LISS-tube or gel IATs with untreated RBCs revealed anti-c in 32 patients with anti-E. When gel-IAT and ficin-pretreated RBCs were used, 21 additional patients with anti-E were found to have anti-c. In samples from 26 R(1)R(1) patients with anti-E, anti-c was not demonstrable by ficin-gel IATs, and in 3 cases, the ficin-gel tests were inconclusive. In five cases in which E- RBCs not tested for c antigen were transfused to patients found by ficin-gel IAT to be without anti-c, all subsequently performed crossmatches with E-, c-untested RBCs were compatible. The incidence of anti-c in R(1)R(1) patients with anti-E in this study was 32 of 82 (39%) with untreated RBCs and 53 of 82 (65%) when the ficin gel data were included. The latter is significantly higher than the 32 percent incidence previously reported (p = 0.0001). Accordingly, all patients at our facility with an Rh antibody are now tested for those additional Rh antibodies they can make, as predicted from their Rh phenotype. The data from this study strongly support the selection of R(1)R(1) RBCs for all c- patients with anti-E.

Apheresis treatment of recurrent focal segmental glomerulosclerosis after kidney transplantation: re-analysis of published case-reports and case-series.

A systematic re-analysis of published cases was performed to better define the role of plasmapheresis in the treatment recurrent focal segmental glomerulosclerosis after renal transplantation. Forty-four cases were identified, of which 32 responded to apheresis. The median number of treatments to response was 9. There was no difference between responders and nonresponders in the total number of treatments performed. The presence of sclerosis on biopsy predicted treatment failure. Relapse after first successful treatment was reported in 10 cases. The median number of treatments received was less and the time from diagnosis to first treatment was greater for patients who relapsed than for patients in whom relapse was not reported, but the differences were not statistically significant. On the basis of this analysis, we recommend early treatment after diagnosis with a regimen of three daily plasmapheresis treatments followed by 6 treatments on an every other day basis.

Revisiting the issue: can the reading for serologic reactivity following 37 degrees C incubation be omitted?

BACKGROUND: Omitting the 37 degrees C reading from screening tests for unexpected antibodies results in failure to detect some Rh, K, and Jk agglutinins of potential significance (wanted positives). However, this measure avoids unwanted positive tests due to cold agglutinins. STUDY DESIGN AND METHODS: Using data from prior publications, actual risk calculations (ARCs) were made to predict the risk of eliminating the 37 degrees C reading, pretransfusion direct antiglobulin test (DAT), and routine indirect antiglobulin crossmatch (IAT-XM). ARCs used the equation: wanted positives missed x 0.34 (or 0.80) x 5 x percent antigen-positive, where 0.34 = percent of patients transfused (ARCs for 37 degrees C reading and DAT); 0.80 = percent of crossmatched patients transfused (ARCs for IAT-XM); 5 = average number of units transfused. Following elimination of the 37 degrees C reading, the impact of this change on patient care was monitored. Antibody detection and identification data and transfusion reaction reports for 6 months after the change were reviewed. Recently transfused patients with new antibodies were evaluated for immune hemolysis by review of clinical and laboratory data. The findings were compared with those from the same dates of the preceding year. RESULTS: The risk of transfusing incompatible blood by eliminating the DAT, IAT-XM, and 37 degrees C reading is approximately 1:13,000, 1:2,000, and 1:2,400 units transfused, respectively. The cumulative risk from eliminating all three tests is approximately. 1 :1,000 units. With respect to the 37 degrees C reading, there were no differences between the pre-change and post-change study periods in the incidence of reported transfusion reactions or cases of immune hemolysis associated with newly formed antibodies. However, unwanted positive tests decreased from 162 to 61 following elimination of the 37 degrees C reading. This represents a decrease of 20 percent in the number of samples requiring antibody identification annually. CONCLUSIONS: Eliminating the 37 degrees C reading from pretransfusion antibody screening tests imposes less risk than omitting the routine IAT-XM, and it avoids the time and costs of evaluating unwanted positive tests, thus reducing expenditures and delays in patient care.

Partial dependence of human peripheral blood leukocyte binding to high molecular weight fucoidan on divalent cations.

L-selectin (CD62L) is the principal leukocyte adhesion molecule for the high endothelial venules of peripheral lymph nodes. This adhesion has an absolute requirement for calcium ions. Nevertheless, some studies have shown carbohydrate adhesion receptor interactions on lymphocytes and neutrophils, including the L-selectin molecule, that are Ca-independent. In the present study fucoidan, a reportedly Ca2+ independent ligand of L-selectin, and Mabs to human CD62L were coupled to magnetic polystyrene beads (MPB), as a model of leukocyte-surface interactions, and the efficiency of human leukocyte separation was investigated. 30% of Ficoll-purified human mononuclear cells and 75% of dextran-purified human leukocytes (DPHL) were specifically bound by fucoidan-modified MPB in the presence of Ca2+; 55% of dextran-purified leukocytes were specifically bound in the absence of Ca2+. The specific binding was inhibited by an excess of free fucoidan. The data obtained show the presence of Ca-independent adhesion determinants, specific to fucoidan on human leukocytes. No significant specific binding of leukocytes to fucoidan-modified MPB was found after the incubation with fresh human Ca(2+)-depleted whole blood. More than 90% of DPHL were specifically bound to MPB modified with Mabs to human CD62L irrespective of Ca2+ presence. The same degree of separation was achieved after the incubation with fresh human Ca(2+)-depleted-whole blood with anti-CD62L modified beads.

Cytokines as intercellular signals in hemolytic transfusion reactions.

Recent clinical and experimental evidence indicates that many of the sequelae of hemolytic transfusion reactions may be mediated by cytokines, including interleukin-1 beta, interleukin-6, tumor necrosis factor-alpha, the chemokines interleukin-8 and monocyte chemoattractant protein-1, and interleukin-1 receptor antagonist. Experimental models of both acute and delayed hemolytic transfusion reactions demonstrate the production of these molecules. The time course and relative patterns of production correlate well with known clinical manifestations of these reactions. Tumor necrosis factor-alpha appears to be central to ABO incompatibility reactions, and stimulates endothelial cells to exhibit procoagulant activity and surface adhesion molecules.

The role of cytokines in hemolytic transfusion reactions.

Experimental evidence is accumulating to support a central role for cytokines in the pathophysiology of hemolytic transfusion reactions. The production of tumor necrosis factor, interleukin-8, and monocyte chemoattractant protein occurs in whole blood in response to ABO incompatible red cells, a model of acute hemolytic transfusion reactions. Peripheral blood mononuclear cells may produce interleukin-1 beta, tumor necrosis factor, interleukin-8, monocyte chemoattractant protein, and interleukin-1 receptor antagonist in response to IgG-coated red cells, a model of delayed hemolytic transfusion reactions. Cultured umbilical vein endothelial cells respond to conditioned plasma from ABO-incompatibility reactions by expressing the procoagulant tissue factor and the leukocyte adhesion molecules ELAM-1 and ICAM-1. These in vitro endothelial cell responses can be inhibited by neutralizing antibodies to tumor necrosis factor, suggesting that TNF may have a central role in intravascular coagulation and end-organ injury that may occur in acute hemolytic transfusion reactions.

White cell-associated procoagulant activity induced by ABO incompatibility.

BACKGROUND: Disseminated intravascular coagulation is an established complication of acute hemolytic transfusion reactions, particularly those involving the ABO red cell (RBC) antigen system. In addition, peripheral blood white cells, particularly monocytes, have demonstrated expression of procoagulant activity (PCA) in response to inflammatory stimuli. To better define the activation of coagulation in immune hemolysis, in vitro experiments were conducted to investigate the expression of PCA by peripheral blood white cells in ABO RBC incompatibility. STUDY DESIGN AND METHODS: Fresh group O heparinized whole blood was incubated with washed, packed group A or O RBCs. White cells were separated, washed, and lysed before assay of PCA, which was measured by a one-stage recalcified clotting time assay. Units of activity were calculated on the basis of a rabbit brain thromboplastin standard curve. Mechanisms of coagulation activation were investigated by using specific coagulation factor-deficient plasmas, blocking antibodies to tissue factor, and anti-CD11b. RESULTS: Significant levels of white cell-associated PCA were found at 2 to 6 hours in response to incompatible (group A) RBCs, but not in response to compatible (group O) RBCs. PCA was not correlated with numbers of platelets in whole blood. Nonimmune lysis of compatible RBCs did not induce PCA. When whole blood reconstituted from washed cells and heat-inactivated plasma was incubated with incompatible RBCs, PCA and hemolysis were abrogated, which suggests that complement is a required intermediate. Protein synthesis inhibition by the addition of cycloheximide (5 mg/mL) to whole blood incubated with RBCs prevented the expression of PCA. Substitution of factor VIII-deficient plasma for normal plasma in the recalcified clotting time assay had no effect, whereas PCA was reduced by 68 percent with factor VII-deficient plasma and was unmeasurable with factor X-deficient plasma. PCA was restored by a 1-to-1 mix of normal and factor VII-deficient plasma. Incubation of samples in the PCA assay with tissue factor antibodies resulted in up to 86-percent inhibition of measured PCA. Titration of the response to the amount of tissue factor antibodies added demonstrated that maximal inhibition occurred with 0.45 mg per mL, above which no further inhibition took place. However, the addition of anti-CD11b (0.75 mg/mL) concomitantly with anti-tissue factor abolished measurable activity. This effect was independent of the amount of added protein, and anti-CD11b alone had no effect on measured activity. The addition to whole blood concomitantly with RBCs of polyclonal antibodies to tumor necrosis factor, sufficient to neutralize 2000 pg per mL, did not alter PCA expression. CONCLUSION: These results indicate that white cell-associated PCA is generated in whole blood in response to ABO RBC incompatibility and may contribute to disseminated intravascular coagulation in acute hemolytic transfusion reactions. Two possible cellular mechanisms are suggested, which involve tissue factor expression and the activation of factor X by a CD11b-dependent mechanism.

In vitro production of interleukin-1 receptor antagonist in IgG-mediated red cell incompatibility.

BACKGROUND: Recently, proinflammatory cytokines including interleukin 1 (IL-1) have been implicated in the pathophysiology of immune-mediated hemolysis. Little is known, however, about the mechanisms by which inflammatory events in these reactions may be downregulated. IL-1 receptor antagonist (IL-1ra) inhibits the actions of IL-1 by competition for cellular receptors, and thus it may regulate the biologic actions of IL-1 during immune-mediated hemolysis. The production of IL-1ra by peripheral blood mononuclear cells (PBMNCs) in response to IgG-coated red cells in vitro was investigated. STUDY DESIGN AND METHODS: Fresh PBMNCs were obtained by density gradient separation of heparinized whole blood. PBMNCs were cultured in the presence of anti-D-coated, Rh-positive red cells or uncoated Rh-negative red cells under nonadherent conditions. IL-1ra concentrations in the culture media were measured by enzyme-linked immunosorbent assay. IL-1ra and IL-1 beta gene expression was assessed by Northern blot analysis of PBMNC mRNA. RESULTS: IL-1ra production was evident 4 hours after stimulation with IgG-coated red cells and increased progressively over 24 hours. Gene expression of IL-1ra was first detected at 2 hours, lagging behind that of IL-1 beta. IL-1ra gene expression was not inhibited by neutralizing polyclonal antibodies to IL-1. Immunocytochemical staining to determine the cellular source localized IL-1ra production to monocytes engaged in erythrophagocytosis. IL-1ra production was inhibited by dexamethasone (10(-7) M). CONCLUSION: These results suggest that IL-1ra production may partly account for the variable pathophysiologic events seen in IgG-mediated autoimmune hemolysis and hemolytic transfusion reactions. Steroid treatment may also downregulate anti-inflammatory cytokine production in immune hemolysis.

Monocyte chemoattractant protein production in red cell incompatibility.

BACKGROUND: Mononuclear phagocytes play a central role in hemolytic transfusion reactions by erythrophagocytosis and production of inflammatory mediators. Factors that affect the number or function of monocyters would be expected to alter the clinical course of hemolytic transfusion reactions, and thus the production of monocyte chemoattractant protein-1 (MCP-1), a recently described chemotactic and activating cytokine specific for monocytes, was investigated in two distinct settings of red cell (RBC) incompatibility. STUDY DESIGN AND METHODS: Fresh heparinized whole blood was incubated with ABO-compatible or -incompatible RBCs. Isolated peripheral blood mononuclear cells were incubated with anti-D-coated or uncoated RBCs. MCP-1 was measured in the plasma or culture medium by enzyme-linked immunosorbent assay. MCP-1 gene expression was detected by Northern blot analysis of buffy coat or mononuclear cell total RNA. RESULTS: Significant levels of MCP-1 protein in plasma or medium were detected 24 hours after the addition of incompatible RBCs, but not in the first 6 hours. Nonimmune hemolysis of added RBCs did not stimulate MCP-1 production. The inactivation of complement by heat treatment of plasma prior to the addition of RBCs to whole blood did not prevent MCP-1 production. Nor did neutralizing antibodies to tumor necrosis factor prevent MCP-1 production in ABO incompatibility. MCP-1 production was associated with increased steady-state levels of white cell MCP-1 mRNA, which occurred more rapidly in ABO than Rh incompatibility. CONCLUSION: The monocyte-specific chemotactic cytokine MCP-1 is produced by peripheral blood leukocytes in response to RBC incompatibility. MCP-1 may act in a positive feedback loop to recruit and activate monocytes during hemolytic transfusion reactions, thus contributing to the maintenance of these reactions.

Molecular genetic analysis of the ABO blood group system: 1. Weak subgroups: A3 and B3 alleles.

We have determined the nucleotide sequences of the coding region in the last two coding exons of ABO genes (which occupy 91% of the soluble form of A1 transferase) from 7 individuals with weak subgroup phenotypes. Four of the individuals had an A3 phenotype and 3 individuals had a B3 phenotype. We determined the nucleotide sequences based on PCR followed by subcloning and DNA sequencing of the amplified fragments. Two cases of the A3 allele and 1 case of the B3 allele were found to contain a single-base substitution which resulted in an amino acid substitution. However, no other cases of A3 and B3 alleles were found to contain differences in this region. This finding demonstrates for the first time heterogeneity among these weak subgroups at the nucleotide level.

Cytokine production in IgG-mediated red cell incompatibility.

The transfusion of incompatible red cells may result in fever and systemic symptoms. The mechanisms by which these symptoms are produced in the setting of antibodies that do not usually fix complement, as in the Rh system, are obscure. It has been hypothesized, on the basis of their known biologic activities, that a specific set of cytokines may be involved in such transfusion reactions. Therefore, the production of the inflammatory cytokines interleukin-1 beta (IL-1 beta), tumor necrosis factor (TNF), interleukin-6 (IL-6), and interleukin-8 (IL-8) by human monocytes in response to red cells sensitized with anti-D was investigated, as a model of IgG-dependent hemolytic transfusion reactions. IL-1 beta, IL-6, and IL-8 were detectable in the culture supernatants at 4 to 6 hours and increased up to 24 hours, whereas TNF peaked at 6 hours. Immunocytochemical stains of cell preparations demonstrated IL-1 beta, IL-8, and TNF in monocytes engaged in erythrophagocytosis. IL-8 production and phagocytosis could be inhibited by monomeric IgG, but Fab fragments of a monoclonal antibody specific for the low-affinity IgG receptor Fc gamma RII could not be, which suggests the involvement of the high-affinity receptor Fc gamma RI. Neutralizing antisera to IL-1 beta and TNF did not abrogate the production of IL-8, which suggests that sensitized red cells serve as a primary signal for this cytokine. These findings indicate that the production of inflammatory cytokines by phagocytes may be responsible for the symptomatology of IgG-mediated hemolytic transfusion reactions.

Anti-i causing acute hemolysis following a negative immediate-spin crossmatch.

A unique case of acute hemolysis following transfusion of red cells (RBCs) that were found compatible by immediate-spin (IS) crossmatch technique is reported. Screening tests for unexpected antibodies, using low-ionic-strength saline (LISS), 10 minutes' incubation at 37 degrees C, and anti-IgG, were nonreactive; however, 1 transfused unit was found crossmatch incompatible by indirect antiglobulin technique (IAT). An anti-i (titer 512 at 4 degrees C) that was not an autoantibody was identified in the patient's serum. Unlike the incriminated donor RBCs, most I+ RBCs did not react by LISS-IAT. Variable reactivity was seen with ficin-treated I+ RBCs, and there was marked hemolysis of iadult and icord RBCs. In marked contrast, dominant Lu(a-b-) RBCs, with reduced expression of i, did not react by any test method; nor did autologous I+, Lu(b+) RBCs. The in vivo clinical significance of this anti-i was confirmed by monocyte monolayer assay and RBC survival studies. The patient's i antigen may have been altered, by either chemotherapy or disease, and lacked part of the i antigen-mosaic. Her antibody was directed at epitopes of i that were absent from her RBCs. Those i epitopes missing from her RBCs are also absent on dominant Lu(a-b-) RBCs. This anti-i represents a unique cause of an acute hemolytic transfusion reaction. It also represents a case of acute immune-mediated hemolysis following transfusion of IS crossmatch-compatible blood when screening tests for unexpected antibodies are nonreactive. Because of the rarity of such cases (less than 1/200,000 RBC units transfused), modifications to pretransfusion testing protocols are not proposed.

Detection of circulating donor white blood cells in patients receiving multiple transfusions.

Significant morbidities are associated with the routine administration of blood products. Although the exact etiology of these complications may be unknown, many are thought to arise from the incidental cotransfusion of "donor" lymphocytes. We have developed an assay to detect small numbers of male white blood cells (WBCs) circulating in female patients who have received multiple blood transfusions using the polymerase chain reaction (PCR). Twenty female patients undergoing major surgical procedures were studied and received an average of 9.3 U of packed red blood cells (4.8 U from male donors) and 11.7 U of platelets (6.1 U from male donors). DNA was extracted from whole blood or peripheral blood buffy coats posttransfusion and PCR performed using oligonucleotides designed to amplify a segment within the repetitive Y-chromosome DYZ1 locus. Posttransfusion, 15 of 20 women showed evidence of circulating male WBCs for an average of 2.0 days (range, 1 to 6). We conclude that (1) DYZ1 PCR analysis is a useful approach for the detection of small numbers of circulating transfused male WBCs in female patients; and (2) circulating donor WBCs persist for a mean of 2.0 days in the majority of women receiving multiple transfusions. Future application of this technique may detect persisting or proliferating WBCs and lead to an improved understanding of common transfusion-related morbidities.

Transfusion management of patients with IgA deficiency and anti-IgA during liver transplantation.

Severe anaphylactic or allergic reactions may occur during blood transfusion to patients who are IgA-deficient and have anti-IgA in their blood, particularly those with class-specific antibodies. These patients are a particular challenge to the hospital transfusion service when large volumes of blood components are required for transfusion support, as in liver transplantation. We have successfully provided blood components for 3 such patients undergoing liver transplantation. Red cells were washed manually or by automated technique. Platelets were washed manually. All plasma was from IgA-deficient donors. One patient's entire plasma requirements were supplied by autologous plasmapheresis. Serial determinations of IgA levels and anti-IgA titers in 1 patient demonstrated an abrupt fall in anti-IgA with the appearance of barely detectable amounts of IgA during the surgery. IgA-containing plasma cells were demonstrated in the biopsies of liver homografts of 2 patients following transplantation. IgA deficiency with anti-IgA can be successfully managed during liver transplantation with advance planning.

Red cell ABO incompatibility and production of tumour necrosis factor-alpha.

Tumour necrosis factor-alpha (TNF) is a major mediator of diverse pathophysiological events similar to those of haemolytic transfusion reactions (HTR), such as fever, intravascular coagulation and organ failure. However, the possible role of TNF in HTR has not been investigated. We have constructed an in vitro whole blood model of HTR to examine whether TNF may be produced in red cell ABO incompatibility. TNF was observed in plasma, in a dose dependent manner, when ABO incompatible red cells were added, but not with compatible (group O) cells. Plasma TNF levels were maximal at 2 h, and declined to control levels by 24 h. Haemolysis of incompatible red cells was accompanied by TNF production. Immune haemolysis induced TNF gene expression by buffy coat leucocytes, as determined by Northern blot analysis. Heat inactivation of plasma abolished TNF production, whereas prior treatment with interferon-gamma augmented the response. These results demonstrate that a major cytokine is produced in response to red cell incompatibility, and suggest that TNF may play a role in the pathogenesis of haemolytic transfusion reactions.

The tall cell variant of papillary carcinoma of the thyroid gland. Comparison with the common form of papillary carcinoma by DNA and morphometric analysis.

The tall cell variant of papillary carcinoma of the thyroid manifests a more aggressive behavior than the usual form of papillary carcinoma of the thyroid. Morphometric analysis of nuclear features and DNA analysis may yield information predictive of aggressive behavior. Accordingly, the DNA content and morphometric features of the neoplastic cells of the tall cell variant were measured and compared with measurements obtained from neoplastic cells of the usual form of papillary carcinoma. Six of the 11 tall cell neoplasms were aneuploid, as were four of the eight usual papillary neoplasms. Although benign cells were separated from malignant cells in each case, differences between tall and usual papillary carcinoma cells were not observed regarding DNA content, chromatin texture, or nuclear size and shape. Differences in the clinical behavior of these neoplasms will likely need to be explained on the basis of other characteristics.