Anti-human neutrophil antigen-1d specificity is frequently observed in anti-human neutrophil antigen-1b alloantisera.

BACKGROUND: Four amino acids are involved in epitope formation of human neutrophil antigens (HNA)-1 alleles, located at positions 36, 65, 78, and 82. HNA-1a and HNA-1b alloantibody epitopes were recently characterized. The HNA-1b allele also carries the HNA-1d epitope p.78A&p.82N. The current study aimed to identify compound antibody specificities in HNA-1b alloantisera, especially the presence of anti-HNA-1d. STUDY DESIGN AND METHODS: For investigation of binding epitopes for HNA-1b alloantibodies, cells stably expressing different HNA-1 alleles were generated and tested against previously well-characterized HNA-1b antisera (n = 11) in an antigen capture assay. Sera with p.82N specificity or p.36S and p.82N specificity were additionally analyzed using adsorption and elution methods. RESULTS: Three amino acids, p.36S, p.78A, and p.82N, are involved in epitope formation of HNA-1b. The following specificities were identified in 11 HNA-1b alloantisera: p.36S (6/11), p.82N (9/11), and p.78A&p.82N (8/11), of which p.36S was identified as a sole entity in 2/11, whereas 9/11 antisera contained a polyspecific mixture of anti-p.36S, p.82N (1/11), and anti-p.78A&p.82N in combination with anti p.82N (5/11) or compound specificities of anti-p.36S, p.82N, and p.78A&p82N (3/11). In seven of eight antisera with p.82N specificity, anti-p.78A&p.82N was detected. DISCUSSION: Analysis of HNA-1b antisera indicates compound specificities for HNA-1b alloantibodies with a high variation between HNA-1b immunized individuals. Amino acids p.36S, p.82N, and p.78A&p.82N are necessary for HNA-1b epitope formation. The HNA-1d epitope is recognized by 73% (8/11) of HNA-1b immunized individuals.

Primary structure of human neutrophil antigens 1a and 1b.

BACKGROUND: Neutrophil specific Fcγ receptor IIIb (CD16b) is a low-affinity IgG receptor. Its polymorphic variants are associated with human neutrophil antigens (HNA). HNA-1a and HNA-1b differ in four amino acids. Immunization can lead to the production of alloantibodies. The exact contribution of four amino acid exchanges for the formation of HNA-1a, -1b epitopes is currently unknown. STUDY DESIGN AND METHODS: Permutation of each polymorphic amino acid from wild-type CD16b cDNA constructs was performed and expressed on HEK293 cells. All 16 receptor variants were produced and tested against 19 well-characterized HNA antisera in an antigen capture assay. RESULTS: Analyzing the reaction pattern revealed that anti-HNA-1a antibodies can bind whenever asparagine (N) is present in position 65, regardless of the three other positions (CD16b *N**). Anti-HNA-1b antibodies can bind when serine (S) is present in position 36 (CD16b S***), when N is present in position 82 (CD16b **N*), or both (CD16b S*N*). CD16b variants with N65 and S36 and/or N82 (such as CD16b SNN*) bind both, anti-HNA-1a and anti-HNA-1b alloantibodies. If these specific amino acids are missing (as in CD16b RSD*), no antibodies will bind. CONCLUSION: Whereas the primary structure of HNA-1a and HNA-1b usually differs in four amino acids, epitope composition is not "antithetical". N65 alone determines the presence of HNA-1a, and S36 and/or N82 determine the presence of HNA-1b. Amino acid 106 does not participate in epitope formation. Our findings are of specific relevance when a HNA-1 phenotype is predicted from a genotype.

Evaluation of a new microbeads assay for granulocyte antibody detection.

BACKGROUND: To reduce the risk of transfusion-associated acute lung injury (TRALI), a high number of plasma donors were tested for human leukocyte antigen (HLA) and human neutrophil antigen (HNA) antibodies. For HNA antibody detection, the gold standard is a combination of the granulocyte immunofluorescence test (GIFT) and the granulocyte agglutination test (GAT). However, these tests are not suitable for a high-throughput of samples. STUDY DESIGN AND METHODS: To evaluate the new generation of the LABScreen MULTI assay (One Lambda, Inc.), which has special new beads for all the known HNA specificities, including HNA-3a, 97 sera samples containing well-defined HNA antibodies were used. For background testing, we used 91 samples from plasma donors previously identified by GAT, GIFT, and the monoclonal antibody-specific immobilization of granulocyte antigens (MAIGA) assay. RESULTS: Compared with previous tests, the new LABScreen MULTI assay was highly specific for the HNA-1a, HNA-1b, HNA-2, and HNA-3a antibody specificities required to prevent TRALI. Ninety-eight percent of the HNA-1a, HNA-1b, and HNA-2 antibodies could be detected as true positive; and 90% of the HNA-3a antibodies were recognized correctly as positive. False-positive reactions were identified in 5.5% of samples that previously tested negative. CONCLUSION: The detection of HNA-3a antibody specificities could be integrated into the new LABScreen MULTI assay; however, we detected only 90%. In addition, we detected further HNA antibodies, such as HNA-1c, HNA-1d, and some HNA-3b and HNA-4a antibodies. The new generation of LABScreen MULTI is a great step toward feasible high-throughput testing for HNA antibodies. Nevertheless, GIFT and GAT remain the gold-standard methods for the differentiation of rare and currently unknown HNA specificities.

Mass spectrometric phosphoproteome analysis of small-sized samples of human neutrophils.

BACKGROUND: Global analysis of stimulus-dependent changes in the neutrophil phosphoproteome will improve the understanding of neutrophil signal transduction and function in diverse disease settings. However, gel-free phosphoproteomics of neutrophils in clinical studies is hampered by limited sample amounts and requires protein extract stability, efficient tryptic digestion and sensitive phosphopeptide enrichment in a protease-rich environment. For development of an appropriate workflow, we assessed neutrophil protein stability in urea-based lysis buffers and determined feasibility of gel-free phosphoproteomic analyses using polymer-based metal ion affinity capture (PolyMAC). METHODS: Western blotting, phosphopeptide enrichment and mass spectrometric analyses of samples of neutrophils were performed. RESULTS: Degradation of proteins in neutrophil extracts was observed after preparation with a urea-containing lysis buffer and could be prevented by addition of highly concentrated protease inhibitors. Subsequent tryptic digestion and PolyMAC-based phosphopeptide enrichment proved efficient with accordingly prepared neutrophil samples. Applying the new workflow, formyl­methionyl­leucyl­phenylalanine-induced phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) was detected after gel-free and gel-based phosphoproteomic analyses as proof of principle from 20 ml of whole blood. Furthermore, phosphorylation of other ERK1/2 pathway-associated proteins was monitored. CONCLUSION: We provide a workflow for efficient, gel-free phosphoproteome analyses with small-sized neutrophil samples, suitable for application in clinical studies.

Human neutrophil antigen-3a antibodies induce neutrophil stiffening and conformational activation of CD11b without shedding of L-selectin.

BACKGROUND: HNA-3a antibodies induce severe transfusion-related acute lung injury (TRALI) in which neutrophils play a major role. As neutrophil passage through the pulmonary microvasculature is a critical step in the pathogenesis of TRALI, we investigated the impact of HNA-3a antibodies on two important factors that could impair granulocyte passage through lung capillaries: the elasticity of neutrophils and the expression and activation of adhesion molecules. STUDY DESIGN AND METHODS: The impact of HNA-3a antibodies on the elasticity of neutrophils was investigated using atomic force microscopy (AFM). Neutrophils were settled on poly-2-hydroxyethyl-methacrylate-coated glass slides before treatment with anti-HNA-3a plasma samples, control plasma, or control plasma containing formyl-methionyl-leucyl-phenylalanine (fMLP). Elasticity measurements were carried out in a temperature-controlled perfusion chamber using an atomic force microscopy (AFM) device. The impact of HNA-3a antibodies on the surface expression of total CD11b, activation of CD11b, and L-selectin (CD62L) shedding was investigated by flow cytometry. The functional impact of HNA-3a antibodies on neutrophil adhesion was assessed using fibrinogen-coated plates. RESULTS: HNA-3a antibodies induced stiffening of neutrophils (+24%-40%; p < 0.05) to a similar extent as fMLP. This effect was blocked by treatment of neutrophils with cytochalasin D. While total surface expression of CD11b and L-selectin on neutrophils was largely unaffected, HNA-3a antibodies induced alloantigen-specific activation of CD11b (+72%-107%; p < 0.05) and increased adhesion of neutrophils to fibrinogen. CONCLUSION: Accumulation of neutrophils in the pulmonary microvasculature during severe TRALI is likely mediated by increased rigidity and CD11b-mediated adhesion of neutrophils leading to retention of neutrophils.

Geno- and phenotyping of human neutrophil antigens.

For typing of human neutrophil antigens (HNA) usually genotyping techniques are used, except for HNA-2, which-due to a gene expression defect-requires phenotyping. For genotyping, several techniques have been described. Most reference laboratories use variations of the polymerase chain reaction (PCR) for antigen typing which showed good results in international quality assessment exercises. The granulocyte immunofluorescence test has been the gold standard technique for phenotyping for all HNA antigens except for HNA-3a and -3b phenotyping. The expression of the latter antigens on neutrophils is often better shown by the use of the granulocyte agglutination test.

Impact of priming on the response of neutrophils to human neutrophil alloantigen-3a antibodies.

BACKGROUND: Human neutrophil alloantigen-3a (HNA-3a) antibodies can induce transfusion-related acute lung injury (TRALI). The severity of TRALI varies largely among the affected patients. Severe comorbidity seems to increase the susceptibility for TRALI, potentially by priming of neutrophils. Thus, the impact of neutrophil priming on HNA-3a antibody-mediated neutrophil aggregation and CD11b surface expression was investigated. STUDY DESIGN AND METHODS: Neutrophils were primed using formyl-methionyl-leucyl-phenylalanine (fMLP) or bacterial lipopolysaccharide (LPS). Granulocyte aggregation and CD11b surface expression were evaluated by the granulocyte agglutination test and by flow cytometry (FC), respectively. Priming-induced changes in the surface expression of choline transporter-like protein 2 (CTL2) and the CTL2 mRNA expression were assessed by FC and quantitative real-time polymerase chain reaction, respectively. RESULTS: Priming of neutrophils lowered the amount of HNA-3a antibodies required for inducing granulocyte aggregation in a dose-dependent manner by 50% to 75%. The priming agent concentration necessary for this response differed between donors. Priming slightly enhanced binding of HNA-3a antibodies to neutrophils. However, CTL2 de novo synthesis was not induced after priming with LPS, indicating that increased HNA-3a antibody binding was likely caused by translocation of intracellular CTL2 to the surface or by increased affinity of HNA-3a antibodies to CTL2. HNA-3a antibodies influenced CD11b surface expression on neutrophils only marginally, which was also not potentiated by priming with fMLP or LPS. CONCLUSION: This study provides experimental evidence supporting the "threshold model" of TRALI. Priming of neutrophils with fMLP or LPS increases their aggregation response to HNA-3a antibodies by lowering the required antibody amount.

The AQP1 mutation c.601delG causes the Co-negative phenotype in four patients belonging to the Romani (Gypsy) ethnic group.

BACKGROUND: The Colton blood group antigens Co(a), Co(b) and Co3 are encoded by the AQP1 gene which produces a water channel forming integral protein. The extremely rare Co-deficiency enables immunisation against the Co3 isoantigen. MATERIALS AND METHODS: Four patients from different regions of Europe who belong to the ethnic minority of Romani (Gypsy) presented with irregular antibodies against a high frequency red blood cell antigen. Positive cross-matches with all red blood cells tested were reported. An Anti-Co3 antibody was identified as the cause of incompatibility in the four cases. The genetic background was determined by polymerase chain reaction typing with sequence-specific primers and by DNA sequencing. RESULTS: The Co(a-b-) phenotype was confirmed in the four patients despite the fact that genotyping revealed the CO*01 allele of the AQP1 gene. A homozygous AQP1 c.601delG mutation, leading to a frame shift and producing a premature stop in the next codon, was responsible for the Co-negative phenotype in all four cases. While one patient was successfully transfused with blood from his sibling with the identical mutation, another case, a baby affected by haemolytic disease of the newborn, recovered without transfusion. DISCUSSION: Despite the difficulties in undertaking a population study to determine the prevalence of this AQP1 c.601delG allele in the ethnic minority of Romani, the observations described in this report clearly suggest an accumulation of this mutation, which causes the Co(a-b-) phenotype, in Romani (Gypsy) patients. Further studies are necessary to prove such an accumulation.

Human neutrophil antigen-3a antibodies induce neutrophil aggregation in a plasma-free medium.

BACKGROUND: Antibodies against the human neutrophil alloantigen-3a (HNA-3a) are involved in severe cases of transfusion-related acute lung injury (TRALI), but the susceptibility of patients towards HNA-3a antibody differs largely. HNA-3a antibodies induce granulocyte aggregation. However, it is unresolved whether plasma proteins are required for granulocyte aggregation. MATERIALS AND METHODS: We investigated whether HNA-3a-antibody-induced aggregation of polymorphonuclear cells is dependent on plasma factors by using and modifying the granulocyte agglutination test (GAT). RESULTS: Polymorphonuclear cells homozygous for HNA-3a did not aggregate when incubated with HNA-3a antibodies in a plasma-protein-free GAT setup. When the GAT was performed using polymorphonuclear cells re-suspended in phosphate-buffered saline containing proteins, HNA-3-mediated aggregation was observed. Moreover, using Tween® 20 for blocking the plates, reconstituted the granulocyte aggregation in a protein-free medium. This indicates that granulocyte aggregation probably occurs by direct granulocyte-granulocyte interaction(s) or is mediated by substances released by neutrophils after activation. DISCUSSION: Granulocyte aggregation induced by HNA-3a antibodies does not require human plasma proteins. Interindividual variability in the response to HNA-3a antibodies does not depend on differences in patient's plasma proteins.

Quality of freeze-dried (lyophilized) quarantined single-donor plasma.

BACKGROUND: Transfusion of plasma is a basic treatment for complex coagulopathies as well as in major blood loss. Early transfusion of plasma after trauma with major hemorrhage has been recommended by retrospective studies. However, the use of plasma is often hampered by the need to maintain a cold chain and the time needed for thawing fresh-frozen plasma (FFP). With freeze-dried (lyophilized) plasma (FDP) both difficulties can be avoided. Here, we describe the production, quality characteristics, and our experiences with FDP. STUDY DESIGN AND METHODS: Quarantine plasma samples were freeze-dried. The clotting factors fibrinogen, Factor (F)V, FVIII, FXI, von Willebrand factor (vWF), protein S, antithrombin, plasminogen, and plasmin inhibitor were determined after manufacturing and after storage at room temperature and refrigeration. Reported adverse transfusion events were evaluated and compared to that of FFP. Clinical effectiveness was estimated by inquiry among experienced users. RESULTS: Lyophilization resulted in a loss of coagulation factor activity between 0% and up to 20% to 25% (FVIII, vWF). When stored refrigerated, coagulation factors did not lose more than 10% of their activities. Storage at room temperature for 24 months mainly affected vWF/ristocetin cofactor activity and fibrinogen activity. From 2007 to 2011 more than 230,000 units of FDP were delivered. There were no reports about clinical ineffectiveness. The frequency of transfusion reactions was not different from that of FFP. CONCLUSION: Lyophilized plasma showed characteristics similar to FFP. Since FDP requires neither complex logistics nor time-consuming thawing, it allows rapid treatment of coagulopathies.

Expression of the CTL2 transcript variants in human peripheral blood cells and human tissues.

BACKGROUND: The HNA-3a antigen is an important antibody target in the pathophysiology of transfusion-related acute lung injury (TRALI). It is encoded by the choline transporter-like protein 2 (CTL2) gene, which exists in the two transcript variants TV1 and TV2, differing in the upstream promoter and coding region. Only TV1 has been demonstrated to enable choline transport across the cell membrane. STUDY DESIGN AND METHODS: The aim of this study was to determine the CTL2 transcript pattern in human peripheral blood cells and tissues and its capacity to bind HNA-3a antibodies. RNA was isolated from human whole blood, isolated neutrophils, mononuclear blood cells, leukoreduced platelets, human lung, liver, and colon. After reverse transcription, the single-stranded cDNA was amplified using primer combinations specific for the respective transcript. Plasmids containing the entire CTL2 coding cDNA of the transcript variant TV1 or TV2 served as controls. HEK293T cells expressing both variants were used to determine the binding of HNA-3a antibodies. RESULTS: The shorter TV2 transcript was demonstrated in each RNA sample derived from human peripheral blood tested so far, as well as in human lung and liver, whereas the longer TV1 transcript was only detected in human lung and colon. TV1 and TV2 had the same binding capacity to HNA-3a antibodies. CONCLUSION: The expression of TV1 and TV2 is tissue and cell specific, with peripheral blood cells expressing only TV2. This does not affect binding of HNA-3a antibodies. Whether the unequal expression might be relevant in the pathogenesis of TRALI remains to be investigated.

Monitoring drug residues in donor blood/plasma samples using LC-(MS)/MS--a pilot study.

Quality assurance of pharmaceutical products is of particular importance and thoroughly controlled. Among these, the preparation of human plasma follows strict guidelines from the point of donor selection to product processing. While various precautions particularly concerning antiviral treatment as well as quality assessment are standard procedure, tests for drug residues are rarely, if at all, conducted with fresh frozen plasma products. With the constantly increasing sensitivity and specificity of modern analytical instruments, the detection of trace amounts of therapeutics in plasma is feasible and can be applied to blood products where considered appropriate. To estimate the prevalence of a selection of commonly prescribed and over-the-counter drugs (including diuretics, beta-receptor blocking agents, contraceptives, ß2 -agonists, antibiotics, antidepressants, analgesics, opioids, glucocorticosteroids, benzodiazepines, stimulants, and oral anti-diabetics) as well as cannabinoids in human donor plasma, a total of 100 specimens (61 female, 39 male) collected at the German Red Cross Organization in 2012 was subjected to an established analytical approach. The methodology was based on protein precipitation followed by liquid chromatographic-high resolution/high accuracy mass spectrometric analysis. Following initial test results, confirmatory analyses were conducted with respective reference substances employing a conventional liquid chromatography-triple-quadrupole mass spectrometer (LC-MS/MS) apparatus. Out of one hundred samples, five were found to contain diuretics (four hydrochlorothiazide and one torasemide), five contained beta-receptor blocking agents (four bisoprolol and one metoprolol), one was found with residues of pseudoephedrine (stimulant) and one with drosperinone (contraceptive). Overall, 12% of samples yielded detectable amounts of drug residues at concentrations estimated to levels common to individuals under therapeutic treatment. In addition, six aliquots of different lots of commercially available plasma preparations with solvent-detergent processing were tested. Here, no drug residues of the targeted therapeutics were detected.

Epitope mapping of antibodies directed against the human neutrophil alloantigen 3a.

BACKGROUND: Severe transfusion-related acute lung injury is often caused by antibodies directed against the human neutrophil alloantigen (HNA)-3a. HNA-3a results from an amino acid exchange (Arg154Gln) in the first extracellular loop of the choline transporter-like protein 2 (CTL2). The characteristics of the binding domain(s) of HNA-3a antibodies are unknown. STUDY DESIGN AND METHODS: For epitope mapping, a library of 23 different HNA-3a (R(154)) and three HNA-3b (Q(154)) peptides covering different parts of the first extracellular loop of CTL2 (aa(55-231)) was synthesized in Escherichia coli and tested by Western blot with two HNA-3a alloantibody-containing plasma samples and by enzyme immunoassay (EIA) with different HNA-3a- (n = 21) and HNA-3b- (n = 1) positive plasma samples. RESULTS: Despite promising Western blot results using highly reactive plasma samples, we found widely varying reactivities of different HNA-3a plasmas in the EIA, with only 11 of 21 HNA-3a antibodies binding to any of the tested HNA-3a peptides and with no peptide recognized by more than nine of the 21 antibodies. The HNA-3b plasma did not react with R(154) peptides in the EIA nor with R(154) or Q(154) peptides in Western blot experiments. Plasma reactivity profiles with the peptides did not correlate with those observed using granulocyte agglutination and granulocyte immunofluorescence tests. CONCLUSION: Binding of HNA-3a alloantibodies depends on the conformation of the intact CTL2 protein and their binding sites may differ substantially. Peptide-based assays for detection of HNA-3a antibodies bear the risk to be insensitive and require systematic validation with a large panel of antibodies.

Genetic variation of the HNA-3a encoding gene.

BACKGROUND: Antibodies against the human neutrophil alloantigen-3a (HNA-3a) play an important role in transfusion-related acute lung injury. The HNA-3a and -3b alloantigens result from a single-nucleotide exchange in the choline transporter-like protein 2 gene (CTL2). We sought for additional polymorphisms that might impair antibody binding to or genotyping of the HNA-3a or -3b antigens. STUDY DESIGN AND METHODS: CTL2-specific complementary DNA (cDNA) fragments were generated from 67 unrelated blood donors followed by DNA sequencing. Polymerase chain reaction with sequence-specific primers (PCR-SSP) was used to test a higher number of donors for relevant new single-nucleotide polymorphisms (SNPs). The granulocyte agglutination test recommended for HNA-3a antibody detection was performed to check HNA-3a antibody binding to the products of the CTL-2 gene variants. RESULTS: Two new missense mutations were demonstrated in the CTL2 cDNA: a 537C>T* exchange leading to a Leu153Phe amino acid substitution and 988C>T variation predicting Thr301Met change. The inherited 537T variant is located in HNA-3a allele results impaired granulocyte agglutination by four of 14 antibodies tested while 988T remains nearly unaffected. CONCLUSIONS: The Leu153Phe exchange next to the HNA-3a/b defining amino acid position can impede the binding of HNA-3a alloantibodies. The HNA-3a genotyping by PCR-SSP might produce misleading results in HNA-3ab heterozygous individuals with the additional CTL2-537T variation of the HNA-3a antigen. These findings must account for the development of new screening assays.

Mechanism of transfusion-related acute lung injury induced by HLA class II antibodies.

Transfusion-related acute lung injury (TRALI) is the leading cause of transfusion-associated mortality in the United States and other countries. In most TRALI cases, human leukocyte antigen (HLA) class II antibodies are detected in implicated donors. However, the corresponding antigens are not present on the cellular key players in TRALI: neutrophils and endothelium. In this study, we identify monocytes as a primary target in HLA class II-induced TRALI. Monocytes become activated when incubated with matched HLA class II antibodies and are capable of activating neutrophils, which, in turn, can induce disturbance of an endothelial barrier. In an ex vivo rodent model, HLA class II antibody-dependent monocyte activation leads to severe pulmonary edema in a relevant period of time, whenever neutrophils are present and the endothelium is preactivated. Our data suggest that in most TRALI cases, monocytes are cellular key players, because HLA class II antibodies induce TRALI by a reaction cascade initiated by monocyte activation. Furthermore, our data support the previous assumption that TRALI pathogenesis follows a threshold model. Having identified the biologic mechanism of HLA class II antibody-induced TRALI, strategies to avoid plasma from immunized donors, such as women with a history of pregnancy, appear to be justified preventive measures.

Characterization of the human neutrophil alloantigen-3a.

Transfusion-related acute lung injury (TRALI) is a frequent cause of transfusion-associated morbidity and mortality. Severe TRALI is often due to antibodies in blood components directed against the human neutrophil alloantigen-3a (HNA-3a). We show here that the HNA-3a antigen arises from a nucleotide polymorphism in the choline transporter-like protein-2 gene (SLC44A2), with the resulting variation at amino acid position 154 determining the reactivity of the protein with HNA-3a-specific antibodies; the variant with an arginine at this position, rather than a glutamine, constitutes the HNA-3a antigen. The molecular identification of this antigen should facilitate the development of assays for blood donor screening to lower the risk of TRALI.

Proteomic characterization of freeze-dried human plasma: providing treatment of bleeding disorders without the need for a cold chain.

BACKGROUND: Transfusion of human plasma is a basic treatment for severe coagulopathies, especially in major bleeding. The required logistics to provide plasma is challenging because of the need to maintain a cold chain. This disadvantage could be overcome by lyophilized plasma. However, it is unknown to what extent lyophilization alters plasma proteins. Quantitative proteomic technologies were applied to monitor protein changes during production of lyophilized, solvent/detergent (S/D)-treated plasma. MATERIALS AND METHODS: The impact of S/D treatment and lyophilization on the plasma proteome was evaluated by differential in-gel electrophoresis (2D-DIGE), and proteins were characterized by mass spectrometry. Clotting factor activities were determined in lyophilized S/D-treated plasma after 24 months of storage at room temperature. RESULTS: By 2D-DIGE, 600 individual protein spots were compared. Lyophilization did not change any of the 600 spots, whereas pathogen inactivation caused significant changes of 38 spots including alpha1-antitrypsin, alpha1-antichymotrypsin, and alpha2-antiplasmin. Clotting factor activities remained stable over 24 months of storage. CONCLUSION: Lyophilization of human plasma neither alters its protein composition nor impairs its clotting capacity. It does not require cost-intensive logistics for storage and transport and can be quickly reconstituted. It is suggested that lyophilized, pathogen-inactivated plasma is an attractive option to provide the most important basic treatment for severe coagulopathies in areas without cold chain and to provide plasma with reduced time delay in emergency situations.