Citrullinated fibrinogen-SAAs complex causes vascular metastagenesis.

Primary tumor cells metastasize to a distant preferred organ. However, the most decisive host factors that determine the precise locations of metastases in cancer patients remain unknown. We have demonstrated that post-translational citrullination of fibrinogen creates a metastatic niche in the vulnerable spots. Pulmonary endothelial cells mediate the citrullination of fibrinogen, changing its conformation, surface charge, and binding properties with serum amyloid A proteins (SAAs), to make it a host tissue-derived metastatic pathogen. The human-specific SAAs-citrullinated fibrinogen (CitFbg) complex recruits cancer cells to form a protein-metastatic cell aggregation in humanized SAA cluster mice. Furthermore, a CitFbg peptide works as a competitive inhibitor to block the homing of metastatic cells into the SAAs-CitFbg sites. The potential metastatic sites in the lungs of patients are clearly visualized by our specific antibody for CitFbg. Thus, CitFbg deposition displays metastatic risks for cancer patients, and the citrullinated peptide is a new type of metastasis inhibitor.

A novel homozygous variant of the thrombomodulin gene causes a hereditary bleeding disorder.

We report a 19-year-old Vietnamese woman who experienced several life-threatening bleeding events, including ovarian hemorrhage. Blood analysis revealed a decreased fibrinogen level with markedly elevated fibrinogen/fibrin degradation products and D-dimer levels. Despite hemostatic surgery and administration of several medications, such as nafamostat mesylate, tranexamic acid, and unfractionated heparin, the coagulation abnormalities were not corrected, and the patient experienced repeated hemorrhagic events. We found that administration of recombinant human thrombomodulin (rhTM) remarkably improved the patient's pathophysiology. Screening and sequencing of the TM gene (THBD) revealed a previously unreported homozygous variation: c.793T>A (p.Cys265Ser). Notably, the Cys265 residue forms 1 of 3 disulfide bonds in the epidermal growth factor (EGF)-like domain 1 of TM. Transient expression experiments using COS-1 cells demonstrated markedly reduced expression of TM-Cys265Ser on the plasma membrane relative to wild-type TM. The TM-Cys265Ser mutant was intracellularly degraded, probably because of EGF-like domain 1 misfolding. The reduced expression of TM on the endothelial cell membrane may be responsible for the disseminated intravascular-coagulation-like symptoms observed in the patient. In summary, we identified a novel TM variant, c.793T>A (p.Cys265Ser). Patients homozygous for this variant may present with severe bleeding events; rhTM should be considered a possible treatment option for these patients.

Screening method for congenital dysfibrinogenemia using clot waveform analysis with the Clauss method.

INTRODUCTION: Congenital fibrinogen disorders (CFDs) are classified as afibrinogenemia or hypofibrinogenemia (Hypo), dysfibrinogenemia (Dys), or hypodysfibrinogenemia (Hypodys), according to functional and antigenic fibrinogen concentrations. However, in routine laboratory tests, plasma fibrinogen levels are mostly measured using the functional Clauss method and not as an antigenic level. Therefore, it is difficult to discriminate CFD from acquired hypofibrinogenemia (aHypo). To establish a screening method for CFD, we investigated the parameters of clot waveform analysis (CWA) from the Clauss method. METHODS: We compared fibrinogen concentrations determined using Clauss and prothrombin time (PT)-derived methods for 67 aHypo and CFD cases (19 Dys, 4 Hypodys, and 1 Hypo determined using antigen levels and DNA sequence analysis) with a CS-2400 instrument, and the CWA parameters, dH and Min1, were analyzed automatically with an on-board algorithm. dH and Min1 are the maximum change in transmittance at the end of coagulation and the maximum velocity of transmittance change during coagulation, respectively. RESULTS: Clauss/PT-derived ratios detected 18 cases of Dys and Hypodys but no Hypo cases, whereas Clauss/dH plus Clauss/Min1 ratios were calculated from fibrinogen concentration using the Clauss method and CWA parameters detected 21 cases of Dys and Hypodys and one Hypo case. Moreover, the Clauss/PT-derived ratio and Clauss/dH plus Clauss/Min1 ratio detected 22 cases of Dys and Hypodys cases and one Hypo case. CONCLUSION: This report demonstrates that CWA parameters of the Clauss method, Clauss/dH plus Clauss/Min1 ratio, screened Dys patients with a higher rate, whereas Clauss/PT-derived ratios did not.

Hereditary Fibrinogen Aα-Chain Amyloidosis in Asia: Clinical and Molecular Characteristics.

Hereditary fibrinogen Aα-chain amyloidosis (Aα-chain amyloidosis) is a type of autosomal dominant systemic amyloidosis caused by mutations in fibrinogen Aα-chain gene (FGA). Patients with Aα-chain amyloidosis have been mainly reported in Western countries but have been rarely reported in Asia, with only five patients with Aα-chain amyloidosis being reported in Korea, China, and Japan. Clinically, the most prominent manifestation in Asian patients with Aα-chain amyloidosis is progressive nephropathy caused by excessive amyloid deposition in the glomeruli, which is similar to that observed in patients with Aα-chain amyloidosis in Western countries. In molecular features in Asian Aα-chain amyloidosis, the most common variant, E526V, was found in only one Chinese kindred, and other four kindred each had a different variant, which have not been identified in other countries. These variants are located in the C-terminal region (amino acid residues 517-555) of mature Aα-chain, which was similar to that observed in patients with Aα-chain amyloidosis in other countries. The precise number of Asian patients with Aα-chain amyloidosis is unclear. However, patients with Aα-chain amyloidosis do exist in Asian countries, and the majority of these patients may be diagnosed with other types of systemic amyloidosis.

Hypodysfibrinogenemia with a Heterozygous Mutation of γCys326Ser by the Novel Transversion of TGT to TCT in a Patient with Pulmonary Thromboembolism and Right Ventricular Thrombus.

We encountered a 45-year-old Japanese man who suffered from pulmonary thromboembolism and huge right ventricular thrombus after inferior vena cava (IVC) filter implantation without apparent thrombus in either the deep veins or inside the IVC filter. The biochemical data showed a discrepancy in the level of fibrinogen between the immunological and thrombin time methods, suggesting hypodysfibrinogenemia. The sequencing of the fibrinogen γ-chain gene (FGG) revealed a novel heterozygous missense mutation in exon 8 - a TGT to TCT transversion in codon 326 - resulting in an amino acid substitution of serine for cysteine (γCys326Ser). The characterization of the protein did not show known mechanisms for thrombosis in dysfibrinogenemia, such as dimer or albumin-binding complex formation. In summary, the current case with a life-threatening thrombotic event was found to have a novel heterozygous missense mutation resulting in γCys326Ser, which was suggested as a predisposing factor of the thrombosis. Known mechanisms responsible for thrombosis in the current case were not demonstrated, suggesting other mechanisms including superimposing inherited and/or acquired risk factors. When a patient presents with unusual thrombosis such as breakthrough pulmonary embolism and huge thrombus in the right ventricle, as in the current case, the laboratory process for heritable thrombophilia should be considered.

[A Case of Secondary Cryofibrinogenemia with Cholangiocarcinoma and Deep Venous Thrombosis].

Cryofibrinogen (CF) is a type of cryoprotein (CP) that can precipitate in cooled plasma but not in serum, and resolves upon warming. We identified a case of secondary cryofibrinogenemia with cholangiocarcinoma and deep venous thrombosis. The patient's cryocrit measured using a Wintrobe tube was 19% in sodium citrate plasma stored for 7 days at 4 degrees C. We performed quantitative analysis of plasma proteins (fibrinogen, IgG, IgA, IgM, C3, C4, α1-antitrypsin, and C-reactive protein) before and after precipitation for 12 hours at 4 degrees C. The plasma fibrinogen concentration decreased by 16.7% (120 mg/dL --> 100 mg/dL), whereas the others were unaffected by precipitation. The CP purified from the patient's plasma was washed three times with saline and subjected to Western blot and Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) analyses. Western blot analysis indicated that the purified CP was composed of not only fibrinogen but also fibronectin, α1-antitrypsin, α2-macroglobulin, coagulation factor VIII, and IgG, IgA, and IgM. Interestingly, SDS-PAGE analysis showed that the molecular weight of the patient's CF differed from that of purified normal fibrinogen (340 KDa) and consisted of several low-molecular-weight bands (50-250 KDa). From these results, we speculated that CF found in this case was a mixture of degradated fibrinogen and some plasma proteins. In summary, cryofibrinogenemia is a rare and under-recognized disease. Sample information in routine clinical practice is valuable to diagnose this disease.

Rapid single nucleotide polymorphism based method for hematopoietic chimerism analysis and monitoring using high-speed droplet allele-specific PCR and allele-specific quantitative PCR.

BACKGROUND: Chimerism analysis is important for the evaluation of engraftment and predicting relapse following hematopoietic stem cell transplantation (HSCT). We developed a chimerism analysis for single nucleotide polymorphisms (SNPs), including rapid screening of the discriminable donor/recipient alleles using droplet allele-specific PCR (droplet-AS-PCR) pre-HSCT and quantitation of recipient DNA using AS-quantitative PCR (AS-qPCR) following HSCT. METHODS: SNP genotyping of 20 donor/recipient pairs via droplet-AS-PCR and the evaluation of the informativity of 5 SNP markers for chimerism analysis were performed. Samples from six follow-up patients were analyzed to assess the chimerism via AS-qPCR. These results were compared with that determined by short tandem repeat PCR (STR-PCR). RESULTS: Droplet-AS-PCR could determine genotypes within 8min. The total informativity using all 5 loci was 95% (19/20). AS-qPCR provided the percentage of recipient DNA in all 6 follow-up patients without influence of the stutter peak or the amplification efficacy, which affected the STR-PCR results. CONCLUSION: The droplet-AS-PCR had an advantage over STR-PCR in terms of rapidity and simplicity for screening before HSCT. Furthermore, AS-qPCR had better accuracy than STR-PCR for quantification of recipient DNA following HSCT. The present chimerism assay compensates for the disadvantages of STR-PCR and is readily performable in clinical laboratories.

Congenital dysfibrinogenemia coincidentally diagnosed at the onset of chronic myelogenous leukemia.

A 34-year-old man was referred to our hospital for leukocytosis and fundal hemorrhage. Peripheral blood and coagulation tests showed increases in cells at all stages of the neutrophilic series and a low level of fibrinogen (Fbg). Chronic myelogenous leukemia (CML) was diagnosed, and nilotinib was administered. During the clinical course of CML treatment, plasma Fbg levels continued to be low, but the patient showed neither hemorrhagic nor thrombotic complications. Fbg analysis showed normal antigen levels and low activity levels, which indicated dysfibrinogenemia. Genetic analysis revealed a heterozygous gene mutation (γ308AAT→AAG), a mutation which was also found in the patient's mother. Asymptomatic patients with dysfibrinogenemia have a low risk of hemorrhage in daily life and do not require treatment. However, in those undergoing major surgery or in serious accidents, replacement therapy may be required. When the cause of low Fbg levels is unknown, dysfibrinogenemia or fibrinogen deficiency should be considered. Even asymptomatic patients may benefit from more detailed immunologic and genetic analyses.

Imaging of thrombosis and microcirculation in mouse lungs of initial melanoma metastasis with in vivo cryotechnique.

Microscopic bioimaging of blood flow and distribution of cancer cells in lungs is essential to analyze mechanism of lung metastasis. Such cancer metastasis has been well known to induce hypercoagulable states and thrombosis. In histopathological tissue sections, however, it has been difficult to capture rapid phenomenon of thrombus formation due to technical problems associated with much less retention of soluble serum components as well as dynamic histological features reflecting their living states. In this study, to achieve bioimaging of both hypercoagulable states and thrombosis induced by early metastasis of mouse B16-BL6 melanoma, "in vivo cryotechnique" (IVCT) was used, which retained soluble components at their original sites. Glutathione-coated quantum dots (QDs) were subsequently injected after melanoma cells via right ventricles to examine plasma flow with fluorescence emission. At 5s after the melanoma injection, melanoma cells were mostly stacked and intruded in alveolar capillaries with changing their shapes. Assembly of platelets initially appeared at 1min, and they aggregated around the stacked melanoma cells at 5min. Such aggregated platelets were immunopositive for both phospho-tyrosine 418 and 527 of Src, indicating their partial signal activation. Fibrin monomers were also immunolocalized around both melanoma cells and platelet aggregates, and massive immunoreaction deposits of fibrinogen were also detected near the same areas, but more strongly detected around the melanoma cells, indicating initial thrombus formation. In those areas, QDs were rarely detected, probably because of the lack of blood supply. Thus, IVCT revealed histopathological features of initial thrombosis under their circulatory conditions.

Nonsense-mediated mRNA decay was demonstrated in two hypofibrinogenemias caused by heterozygous nonsense mutations of FGG, Shizuoka III and Kanazawa II.

We report two novel hypofibrinogenemias, Shizuoka III and Kanazawa II, which are caused by heterozygous mutations in FGG. Shizuoka III showed c.147delT and 147_149insACA in FGG exon 3 and a subsequent frameshift mutation, resulting in mature protein γ23X (native protein: γ49X), and Kanazawa II showed c.1205G>A in FGG exon 9, resulting in γ376X (native protein: γ402X). To determine whether the truncated γ-chains, γ23X and γ376X, were synthesized and participated in the assembly of fibrinogen, mutant-type cDNA vectors were transfected into Chinese hamster ovary (CHO) cells. Significant levels of mutant fibrinogen were not detected by ELISA in the culture media and cell lysates. Immunoblot analysis of cell lysates revealed that the mutant γ-chain of γ376X was observed but intact fibrinogen was not. On the other hand, mutant γ-chain was not observed in γ23X-expressing cells. To demonstrate the involvement of the mechanisms of nonsense-mediated mRNA decay (NMD), we cloned wild- and mutant-type mini-genes containing γ23 or γ376 codon and transfected these into CHO cell lines in the absence or presence of cycloheximide as an NMD inhibitor. mRNA levels were determined using real-time quantitative RT-PCR in CHO cells. In the absence of cycloheximide, levels of mRNAs transcribed from the mutant gene were lower than from the wild-type gene whereas, in the presence of cycloheximide, levels of mRNAs transcribed from the mutant gene increased dose-dependently. Finally, these results demonstrated that mRNAs containing γ23X or γ376X are degraded by the NMD system and translation of the truncated γ-chain polypeptide decrease in patients' hepatocytes, resulting in hypofibrinogenemias.

Successful living-related kidney transplantation in a boy with inherited dysfibrinogenemia.

In kidney transplantation, it is essential to avoid acute vascular complications, such as hemorrhage and renal vascular thrombosis, which may often lead to allograft loss. Inherited dysfibrinogenemia is a rare coagulation disorder with a wide spectrum of clinical manifestations, such as excessive bleeding and thrombosis. A 12-yr-old boy, previously diagnosed with renal hypodysplasia, was found to have reduced fibrinogen concentrations. Coagulation tests assessing surgical risk during kidney transplantation showed a discrepancy between functional and immunologic fibrinogen concentrations. Gene analysis confirmed inherited dysfibrinogenemia, with a heterozygous mutation in FGA (Aα Arg16His) in the patient and his mother. Based on the molecular and functional properties of the mutation, and a familial phenotype, in which his aunt had experienced a previous bleeding episode, the patient was considered at greater risk of bleeding than of thrombosis. The patient was administered fibrinogen concentrate before surgery, and kidney transplantation was performed with his father as the organ donor. The patient received additional prophylactic infusions of fibrinogen concentrate postoperatively, and his postoperative course was uneventful. Accurate diagnosis of dysfibrinogenemia, including gene analysis, is important for correctly managing patients with this coagulation disorder who are undergoing kidney transplantation.

siRNA down-regulation of FGA mRNA in HepG2 cells demonstrated that heterozygous abnormality of the Aα-chain gene does not affect the plasma fibrinogen level.

INTRODUCTION: We encountered two afibrinogenemia patients with homozygous and compound heterozygous FGA mutation. Of interest, the patients' parents, who are heterozygous, had normal levels of plasma fibrinogen; thus, we hypothesized that liver FGA mRNA levels were higher than those of FGB and/or FGG mRNA. MATERIALS AND METHODS: To test the hypothesis, we quantitated mRNA levels of a normal liver and a human hepatocyte cell line, HepG2 cells, and performed siRNA-mediated down-regulation of the fibrinogen gene in HepG2 cells. mRNA levels were determined using real-time quantitative RT- PCR for three normal livers and HepG2 cells. Down-regulation of FGA, FGB, or FGG in HepG2 cells was performed by the addition of siRNA corresponding to each of the three genes, and the mRNA levels determined in the cells and the secreted fibrinogen concentration in media. RESULTS: The mRNA level of normal human liver was FGA=FGB>FGG and the FGG mRNA level was about 2-fold lower than the others, that of HepG2 cells was FGA>FGG>FGB and FGA mRNA was approximately 2- or 4-fold higher than FGG mRNA and FGB mRNA. When FGA, FGB, or FGG mRNA expression levels were down-regulated by nearby 50%, fibrinogen concentrations in media were 78%, 49%, or 57% of the control, respectively. CONCLUSIONS: Our results suggest that FGG mRNA levels limit fibrinogen expression in normal liver and HepG2 cells and that 50% reduction of FGA mRNA levels would not limit fibrinogen expression in normal liver and HepG2 cells.

Waldenström macroglobulinemia transforming from t (11;18) (q21;q21) -negative gastric MALT lymphoma after systemic dissemination.

AIM: We here describe the clinical course of a 70-year-old male patient with Waldenström macroglobulinemia (WM) putatively transformed from refractory mucosa-associated lymphoid tissue lymphoma (MALTL). METHODS: Immunological staining was performed on formalin-fixed, paraffin-embedded tissue sections, and M-protein and cryoglobulin were identified by immunofixation electrophoresis and the cold precipitation method. Chromosome translocation was analyzed by the G-banded karyotype, and API2/MALT1 fusion gene underwent fluorescent in situ hybridization. Multiplex polymerase chain reaction was performed to analyze the VH-JH or DH-JH rearrangements of the IGH gene. RESULTS: At diagnosis, the WM patient had monoclonal IgM with cryoglobulinemia and hyperviscosity syndrome. Eight years before developing WM, the patient experienced the onset of typical gastric MALT-L with H. pylori infection, but in spite of negative for chromosome translocation, t (11;18) and the successful eradication of H. pylori, the MALT-L relapsed repeatedly, and finally led to systemic metastasis. The lymphoma cells also infiltrated the large intestine and spleen. Immunoglobulin gene analyses of cellular clonality revealed that the same clone had been present in the stomach, bone marrow (BM) at the onset of MALT L, and in the BM at the diagnosis of WM. CONCLUSIONS: In this case, lymphoma developed as H. pylori-associated gastric MALT-L with negative for t (11;18), and might be transformed into MW during the systemic metastasis.

Application of allele-specific quantitative PCR using genomic DNA to monitor minimal residual disease based on mutant gene levels following allogeneic hematopoietic stem cell transplantation in patients with hematological malignancies: comparison of mutant levels with autologous DNA percentage by short tandem repeat-PCR.

BACKGROUND: Quantitative evaluation of minimal residual disease (MRD) following hematopoietic stem cell transplantation (HSCT) is indispensable for patients with hematological malignancies. In addition to established MRD markers such as immunoglobulin and T-cell receptor gene rearrangements, fusion genes, or aberrantly expressed genes, single nucleotide mutations are considered one of the MRD markers that reflect the malignant cell clone. METHODS: We compared the quantity of mutant genes by allele-specific quantitative polymerase chain reaction (AS-qPCR) for single nucleotide mutations (TP53 410T>A and PTPN11 1508G>A) with the percentage of autologous DNA by short tandem repeat (STR)-PCR. RESULTS: Following HSCT, the quantity of mutant genes detected by AS-qPCR correlated with the percentage of autologous DNA assessed by the STR-PCR. Moreover, mutant DNAs were detected at a quantifiable level before relapse, whereas the percentage of autologous DNA was less than 5%, that is, complete chimerism. CONCLUSIONS: The AS-qPCR approach for single nucleotide mutations was accurate and highly sensitive for monitoring pre-transplantation as well as post-transplantation MRD. AS-qPCR for single nucleotide mutation is suitable for monitoring MRD in patients who lack previously established MRD markers.

Quantitative monitoring of single nucleotide mutations by allele-specific quantitative PCR can be used for the assessment of minimal residual disease in patients with hematological malignancies throughout their clinical course.

BACKGROUND: Monitoring of minimal residual disease (MRD) in patients with hematological malignancies is important for evaluating the patients' therapeutic response and risk of relapse. Single nucleotide mutations associated with leukemogenesis can be considered as applicable MRD markers. METHODS: We developed an allele-specific quantitative polymerase chain reaction (AS-qPCR) for FLT3 2503G>T, KIT 2446G>T, and KIT 2447A>T and compared the change in the expression levels of the FLT3 or KIT mutations assessed by AS-qPCR to those of the RUNX1-RUNX1T1 fusion gene and WT1 by conventional quantitative PCR. RESULTS: The AS-qPCR using primers including template-mismatched nucleotide or template-mismatched nucleotide plus locked nucleic acid substituted nucleotide provided higher selectivity for mutant nucleotides. The change in the expression levels of the FLT3 or KIT mutations at the time of relapse and just after hematopoietic stem cell transplantation correlated well with that of the RUNX1-RUNX1T1 fusion gene and WT1. Moreover, during complete remission, only AS-qPCR could detect low-level expression of residual mutations. CONCLUSIONS: The AS-qPCR for analyzing single nucleotide mutations contributes to the monitoring of MRD in patients without recurrent fusion gene throughout the clinical course and thus broadens the spectrum of patients in whom MRD can be monitored.

Characterization of cysteine and homocysteine bound to human serum transthyretin.

BACKGROUND: Cysteine-bound transthyretin (TTR) in serum is believed to be associated with deposition of amyloid in familial amyloidotic polyneuropathy (FAP). We examined the binding of cysteine and homocysteine with TTR, and the effect of such binding on TTR structure. METHODS: The concentration of serum cysteine and homocysteine was measured using HPLC and fluorescence detection. TTR was analyzed using isoelectric focusing (IEF), SDS-polyacrylamide gel electrophoresis (PAGE) and mass spectrometry. RESULTS: Incubation of serum at 4 degrees C resulted in an approximate doubling of the amount of both cysteine and homocysteine bound to TTR. Incubation of serum TTR with cysteine-HCl at 37 degrees C markedly altered the IEF and SDS-PAGE profiles of TTR. The main ion peaks of TTR were observed at m/z 13,761 and 13,881, and assigned as free TTR and TTR+cysteine respectively. Incubation of purified TTR with dithiothreitol followed by cysteine-HCl resulted in loss of the latter peak and an increase in the m/z 13,761 peak, while incubation with cysteine-HCl alone did not cause such a change. CONCLUSION: Cysteine- and homocysteine-bound TTR was more easily denatured by cysteine-HCl than free TTR. Binding of cysteine and homocysteine may alter the structure and characteristics of serum TTR, and may facilitate TTR denaturation and deposition.

Residue gamma153Cys is essential for the formation of the complexes Aalphagamma and Bbetagamma, assembly intermediates for the AalphaBbetagamma complex and intact fibrinogen.

BACKGROUND: Fibrinogen Matsumoto IV was found in a hypofibrinogenemia caused by a heterozygous missense mutation, i.e., the substitution of the fibrinogen gamma-chain residue Cys153 by Arg. METHODS: To examine the precise basis for the fibrinogen deficiency, mixtures of any two vectors, the fibrinogen Aalpha-, Bbeta-, gamma- (153Cys) or gammam-(153Ala) chain were transfected into Chinese hamster ovary cells (CHO-Aalpha/gamma, -Aalpha/gammam, -Bbeta/gamma, -Bbeta/gammam). Expression and constitution of each of two chains and their complexes in the individual CHO cell lines were identified by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and immunoblot analysis using polypeptide specific antibodies and two-dimensional electrophoresis (2-DE). RESULTS: In the CHO-Aalpha/gamma and -Bbeta/gamma, the Aalpha/gamma- or Bbeta/gamma-complex was formed, whereas in the CHO-Aalpha/gammam and -Bbeta/gammam, no Aalpha/gammam- or Bbeta/gammam-complex was observed. These results demonstrate that gamma153Ala cannot assemble with the Aalpha- and Bbeta-chains, leading to impaired fibrinogen assembly and secretion. CONCLUSION: gamma153Cys is an essential residue for the fibrinogen assembly which is dependent on Aalpha/gamma- and Bbeta/gamma-complex formation.

Immunohistochemical localization of sodium-potassium ATPase in human normal stomach and gastric adenocarcinomas.

We studied the expression pattern of Na, K-ATPase beta 1 subunit in human normal stomachs and in gastric adenocarcinomas by using anti-Na, K-ATPase beta 1 subunit-specific monoclonal antibody. Tissue samples were processed in formalin solution or in a cold acetic acid-ethanol solution, routinely processed, embedded in paraffin, and an immunoperoxidase method for Na, K-ATPase beta 1 subunit was performed. After antigen retrieval using a steamer in citrate buffer (pH 6.0), tissue sections initially fixed in cold acetic acid-ethanol showed intense immunoreactivity with the antibody at the lateral or basolateral cytoplasmic membrane of normal gastric epithelial cells, at the cytoplasmic membrane of gastric carcinoma cells according to the level of differentiation, and at the cytoplasmic membrane and in the cytoplasm of Schwann cells and neurons in the mesenteric plexus of the gastric wall. Acetic acid-ethanol and paraffin embedding is a useful method for the investigation of the immunohistochemical localization of Na, K-ATPase in normal and diseased tissues.