Delta-Hemoglobin Equivalent and Granularity Index as Cell-Derived Biomarkers for the Detection of Bacterial Infections.

BACKGROUND: Prompt and precise detection of an infection in the blood is of great clinical importance in terms of early therapy initiation and the patient's prognosis. Infection-triggered inflammatory cellular and humoral signaling cascades offer great opportunities to redefine standard tests. However, while inexpensive and easy-to-use biomarkers for the detection of infections and the concomitant inflammatory processes exist, they are rarely used in clinical practice. We aimed to investigate the correlation of Granularity Index (GI) and Delta-hemoglobin equivalent (Delta-He) as inexpensive and easy-to-use cell-derived infection markers with established acute-phase parameters in a randomly selected patient. METHODS: We analyzed plasma concentrations of the established C-reactive protein (CRP) and procalcitonin (PCT) and leukocyte and thrombocyte counts in blood samples of 1,787 patients undergoing routine laboratory inflammation diagnostics. We also measured the Granularity Index (GI) and Delta-hemoglobin equivalent (Delta-He) in this cohort between February 2019 and February 2020. RESULTS: Delta-He and GI Index significantly correlated with CRP concentration (AUC 0.72, 95% CI 0.71 - 0.74; p < 0.001 for both analytes) and thrombocyte count (p < 0.001 for both analytes) but not with leukocyte count (AUC 0.54, 95% CI 0.50 - 0.59, p < 0.67). Furthermore, Delta-He significantly correlated with PCT (AUC 0.65, 95% CI 0.63 - 0.68, p < 0.001) while GI Index did not. Additionally, thrombocyte count significantly correlated with CRP (p < 0.001) and with PCT concentrations (p < 0.001). CONCLUSIONS: Delta-He and GI are two novel, inexpensive and easy-to-use cell-derived hematological biomarkers with the potential to be used as fully automated and highly standardized parameters. These biomarkers would be available on a 24 hours basis in the routine laboratory for the detection of bacterial infections by measuring a complete blood count (CBC) with differential and reticulocyte counts.

Harmless Pregnancy-Induced Warm Autoantibodies to Red Blood Cells.

BACKGROUND: There is little information concerning the development and significance of autoantibodies (aab) to red blood cells (RBCs) during pregnancy. METHODS: Unselected pregnant women were routinely screened for the presence of unexpected antibodies to RBCs using standard techniques. RESULTS: Between 2009 and 2013, 153,612 pregnant women were tested. The antibody screening test was positive in 1,721 women (1.12%). In 1,602 (1.04%) cases, immune and/or non-immune alloantibodies and cold-reactive aab were detected, whereas warm-reactive aab were found in 119 women (0.08%). In almost all cases, warm-reactive aab belonged to the IgG class. No evidence of the presence of significant haemolysis in affected women was observed. CONCLUSION: Pregnant women may rarely develop aab to RBCs, which do not appear to cause haemolytic anaemia. Further clarification is required on the reasons behind the development of these aab and their clinical insignificance.

Evidence Suggesting Complement Activation and Haemolysis at Core Temperature in Patients with Cold Autoimmune Haemolytic Anaemia.

BACKGROUND: It is unclear why haemolysis may somewhat persist in patients with cold autoimmune haemolytic anaemia (cAIHA) at 37 °C (core temperature). METHODS: Seven patients with cAIHA were included in this study. Serological testing was performed using standard techniques. Bound autoantibodies (aab) on patients' RBCs were analysed by the direct antiglobulin test (DAT), dual antiglobulin test (DDAT) and flow cytometry (FC) using pre-warmed RBCs (37 °C). Temperature-dependent complement binding was determined by incubation of patients' serum samples with group O RBCs and fresh serum complement. RESULTS: The DAT was strongly positive with anti-C3d in all cases, independent of season and outside temperature. Haemolysis usually improved during warm periods of time, but decompensated following febrile infections, and persisted throughout the year, though exposure to the cold was strictly avoided. In addition, trace amounts of IgM aab were infrequently detectable on patients' RBCs even at 37 °C, and complement activation was demonstrated following incubation of RBCs with the causative aab at 37 °C. CONCLUSIONS: Binding of trace amounts of IgM aab at 37 °C may provide an explanation for the durable C3d-positive DAT and haemolysis in patients with cAIHA.

Variability of Findings in Drug-Induced Immune Haemolytic Anaemia: Experience over 20 Years in a Single Centre.

BACKGROUND: Drug-induced immune haemolytic anaemia (DIHA) is difficult to diagnose, and its true incidence remains obscure. Here, we present cases of DIHA identified at our institute over the last two decades. METHODS: Serological tests were performed according to standard procedures. Detection of drug-dependent antibodies was performed in the presence and absence of the relevant drug and/or their ex vivo antigens. RESULTS: Over the last 20 years, 73 patients have been identified with DIHA in our institute, which was related to 15 different drugs. The most common single drugs identified were diclofenac (n = 23), piperacillin (n = 13), ceftriaxone (n = 12) and oxaliplatin (n = 10). As far as data were available, haemolysis was acute in all patients, and signs of intravascular haemolysis were present in 90% of the cases. Haemolysis resulted in death in 17 patients (23%). The remaining patients recovered, but haemolysis was complicated by transitory renal and/or liver failure or shock in 11 patients. Upon initial evaluation, the antibody screening test was positive in 36 cases. A positive direct antiglobulin test (DAT) at least with anti-C3d was found in 65 cases, with anti-IgG only in 6 cases, and with anti-IgA only in 1 case. CONCLUSION: DIHA is a rare but potentially life-threatening disorder that should be considered if a patient develops haemolysis under drug treatment. The main serological finding is a positive DAT, primarily with anti-C3d.

Precautions surrounding blood transfusion in autoimmune haemolytic anaemias are overestimated.

BACKGROUND: It is very evident that many precautions are taken regarding transfusion of red blood cells in patients with autoimmune haemolytic anaemia. Frequently, considerable efforts are made to examine the indication and serological compatibility prior to transfusion in such patients. However, at times, this may unnecessarily jeopardize patients who urgently require a red blood cell transfusion. MATERIALS AND METHODS: Thirty-six patients with warm-type autoimmune haemolytic anaemia were included in this study. All patients had reactive serum autoantibodies and required blood transfusion. Standard serological assays were employed for the detection and characterization of antibodies to red blood cells. RESULTS: A positive direct antiglobulin test was observed in all 36 patients, in addition to detectable antibodies in both the eluate and serum. Significant alloantibodies were detected in the serum samples of three patients (anti-c, anti-JK(a), and anti-E). In 32 patients, red blood cell transfusion was administered with no significant haemolytic transfusion reactions due to auto- and/or allo-antibodies. Due to overestimation of positive cross-matches three patients received no transfusion or delayed transfusion and died, and one patient died due to unrecognised blood loss and anaemia which was attributed to an ineffective red blood cell transfusion. DISCUSSION: Many of the reported recommendations regarding transfusion of red blood cells in autoimmune haemolytic anaemia are highly questionable, and positive serological cross-matches should not result in a delay or refusal of necessary blood transfusions.

Angiotensin converting enzyme intron 16 insertion/deletion genotype is associated with plasma C-reactive protein concentration in uteroplacental dysfunction.

INTRODUCTION: Disturbance of the uteroplacental circulation (UPC) and the renin-angiotensin system are involved in the pathogenesis of preeclampsia. In women with history of preeclampsia persistently elevated C-reactive protein (CRP) levels have been described. The angiotensin-converting enzyme (ACE) intron 16 insertion/deletion (I/D) genotype is associated with ACE activity and assumed to be a risk factor for preeclampsia. As ACE generates proinflammatory angiotensin II, we analysed, whether ACE intron 16 I/D genotype is associated with CRP and whether this association exhibited a relation to uteroplacental dysfunction. MATERIALS AND METHODS: A total of 639 women have been followed during pregnancy with repeated measurements of CRP levels (observations: n=2333). ACE intron 16 I/D genotype was determined, and its association with CRP was assessed with adjustment for non-independent observations. RESULTS: CRP levels of ACE D allele carriers were significantly higher than those of the ACE II (wild-type) genotype (p=0.0003, p(adj)=0.04). This relation was allele-dose dependent (p<10(-4), p(adj)<0.02). Association between ACE I/D and CRP was significantly restricted to patients presenting with impaired UPC in univariate (p<0.04) and multivariate analyses (p=0.01). CONCLUSIONS: The ACE I/D genotype is significantly associated with CRP elevations during pregnancies complicated by disturbed UPC. Whether this effect on CRP is involved in pathogenesis of preeclampsia has to be elucidated.

Intravenous artesunate for severe malaria in travelers, Europe.

Multicenter trials in Southeast Asia have shown better survival rates among patients with severe malaria, particularly those with high parasitemia levels, treated with intravenous (IV) artesunate than among those treated with quinine. In Europe, quinine is still the primary treatment for severe malaria. We conducted a retrospective analysis for 25 travelers with severe malaria who returned from malaria-endemic regions and were treated at 7 centers in Europe. All patients survived. Treatment with IV artesunate rapidly reduced parasitemia levels. In 6 patients at 5 treatment centers, a self-limiting episode of unexplained hemolysis occurred after reduction of parasitemia levels. Five patients required a blood transfusion. Patients with posttreatment hemolysis had received higher doses of IV artesunate than patients without hemolysis. IV artesunate was an effective alternative to quinine for treatment of malaria patients in Europe. Patients should be monitored for signs of hemolysis, especially after parasitologic cure.

Piperacillin-induced immune hemolysis: new cases and a concise review of the literature.

BACKGROUND: In this study, the data of eight new patients in conjunction with previously reported cases with piperacillin-induced immune hemolytic anemia (PIHA) are described. STUDY DESIGN AND METHODS: Five patients with cystic fibrosis and three patients with other disorders developed massive hemolysis after administration of piperacillin. Serologic tests were carried out using standard techniques. Tests for drug-dependent antibodies (ddab) were performed in the presence and absence of piperacillin and its ex vivo antigens (urine of patients treated with piperacillin). RESULTS: Hemolysis was acute and severe in all eight patients. The direct antiglobulin test was positive in all cases. Sera from four patients reacted with red blood cells (RBCs) in the presence of piperacillin as well as its ex vivo antigens. Sera from three patients showed positive reactivity with untreated RBCs in the presence of piperacillin, and the serum from the remaining patient was reactive only in the presence of piperacillin ex vivo antigens. Other patients with PIHA have been reported in the literature. Three of these patients also had cystic fibrosis. CONCLUSION: To date approximately 26 patients with PIHA have been described and at least eight of these patients had cystic fibrosis. It is unclear whether patients with cystic fibrosis might be susceptible in developing PIHA.

New antigen in the Dombrock blood group system, DOYA, ablates expression of Do(a) and weakens expression of Hy, Jo(a), and Gy(a) antigens.

BACKGROUND: The Dombrock (Do) blood group system consists of five distinct antigens: Do(a), Do(b), Gy(a), Hy, and Jo(a). Our finding of a patient whose plasma contained a Do-related alloantibody suggested the presence of a sixth antigen. STUDY DESIGN AND METHODS: Standard hemagglutination, flow cytometry, and polymerase chain reaction (PCR)-based methods were used throughout. Protein homology modeling was used to map the amino acid change on the protein structure. RESULTS: The patient's red blood cells (RBCs) typed as Do(a-b-), Hy+(w), Jo(a+(w)), and Gy(a+(w)). The patient's plasma agglutinated RBCs with common Dombrock phenotypes. Reactivity with Hy- and Jo(a-) RBC samples was weak, and Gy(a-) RBC samples were nonreactive. DNA analysis showed the patient to be DO*793A (DO*A/DO*A), DO*323G, and DO*350C, which predicts the Do(a+b-), Hy+, and Jo(a+) phenotype, and revealed a homozygous single-nucleotide change of 547T>G in Exon 2 that is predicted to change tyrosine at Amino Acid Position 183 to aspartic acid. This missense substitution introduced a BtgZI restriction enzyme site. The sequence data were confirmed with a PCR-restriction fragment length polymorphism assay and revealed that the patient's parents and children were heterozygous DO*547T/G. Homology modeling predicted that the 183Tyr substitution by Asp altered the Cys182 environment and influenced the formation and/or stability of the Cys182-Cys231 disulfide bond. CONCLUSION: The patient's DO genes have a single-nucleotide change, which leads to the absence of the high-prevalence antigen DOYA. The absence of this antigen is associated with 183Asp and silencing of Do(a) and weakening of Gy(a), Hy, and Jo(a) antigens.

Mixed-type autoimmune hemolytic anemia: differential diagnosis and a critical review of reported cases.

BACKGROUND: Autoimmune hemolytic anemia (AIHA) is usually classified as either warm or cold type. During the past few decades, mixed types (Mxs) have also been described in a number of cases (6%-8% of AIHA), often without serologic data to support the diagnosis. In this study, we demonstrate that the incidence of Mx AIHA in our institution is extremely rare. STUDY DESIGN AND METHODS: Between August 1998 and August 2007, all in- and outpatients with detectable warm autoantibodies (WABs) were included in this study. Serologic testing was performed using standard techniques for the detection of red blood cell antibodies. RESULTS: From a total of 2194 patients with detectable WABs, only 2 patients (<0.1%) developed both WABs and cold agglutinins (CAs), which in the presence of clinical evidence of hemolytic anemia, satisfies the criteria for Mx AIHA. Only 1 of these patients, however, showed cold and warm hemolysis. Insignificant CAs at temperatures of not more than 24 degrees C were found in 242 patients. CONCLUSION: There is evidence that the presence of CAs with high thermal amplitude and WABs may lead to confusion and misdiagnosis in some patients with AIHA. This study demonstrates that Mx AIHA is less common than previously reported.