Development of antibody against epitope of lipoprotein(a) modified by oxidation: evaluation of new enzyme-linked immunosorbent assay for oxidized lipoprotein(a).

BACKGROUND: Recently, the biological effects of oxidized lipoprotein(a) [Lp(a)] have been reported to be more potent than Lp(a), the arteriosclerosis-relevant lipoprotein. Thus, investigations with oxidized Lp(a) are expected to provide viewpoints different from the conventional ones based on Lp(a). METHODS AND RESULTS: An anti-Lp(a) monoclonal antibody (161E2) was produced against synthetic peptide antigen (Arg-Asn-Pro-Asp-Val-Ala-Pro). This epitope was characterized as having various properties because its external exposure was induced as a result of oxidative modification. Using 161E2 antibody, we developed a new enzyme-linked immunosorbent assay to measure Lp(a) modified by oxidative stress. The present data demonstrated that oxidized Lp(a) that contains the epitope of 161E2 antibody was present in the serum of humans. Therefore, we used this new enzyme-linked immunosorbent assay to evaluate the role of oxidized Lp(a) in patients with hypertension, which induces oxidative stress. Interestingly, hypertensive patients with complications showed a significantly higher level of oxidized Lp(a) in serum than did normotensive subjects (P:<0.01), whereas there was no significant difference in native Lp(a) between normotensive and hypertensive subjects. Importantly, positive immunostaining with 161E2 monoclonal antibody was found in the human arteriosclerotic tissue. CONCLUSIONS: We developed a new antibody against an epitope in Lp(a) as a result of oxidation treatment but not in native Lp(a). The present data demonstrated in vivo the presence of oxidized Lp(a) in the atherosclerotic tissue and its elevation in hypertensive patients. The presence of oxidized Lp(a) may be important in understanding the role of Lp(a) in cardiovascular disease.

Conditioned medium from HepG2 cells transfected with human apolipoprotein(a) gene stimulates growth of human vascular smooth muscle cells: effects of overexpression of human apolipoprotein(a) gene.

Lipoprotein(a) [Lp(a)] is well known to stimulate growth of vascular smooth muscle cells (VSMCs), resulting in atherosclerosis. Its mechanism is postulated to be decreased in active transforming growth factor (TGF)-beta. However, the exact mechanisms and cellular processing from apolipoprotein(a) [apo(a)] to Lp(a) have not yet been clarified because no cultured cells producing apo(a) are available. Therefore, it is necessary to establish apo(a)-producing cells to study the role of apo(a). We evaluated the effects of overexpression of human apo(a) gene on human aortic VSMC growth. First, we tested whether transfection of apo(a) gene into human hepatoma cells, HepG2 cells, producing human apoB resulted in the formation of Lp(a). Transfection of apo(a) gene into HepG2 cells resulted in detectable levels of Lp(a) in the medium, as assessed by ELISA and Western blot, whereas no Lp(a) was detected in the medium of HepG2 cells transfected with control vector and untransfected HepG2 cells. Expression of apo(a) mRNA was also confirmed by reverse transcription-polymerase chain reaction. In contrast, Western blotting showed a single band detected by specific anti-apo(a) antibody, but not anti-apoB antibody, in the medium of apo(a)-transfected VSMCs. These results demonstrate that Lp(a) can be formed from apo(a) on HepG2 cells, whereas transfection of apo(a) gene into VSMCs resulted in the production of apo(a) alone but not Lp(a). Next, we examined the biological effects of overexpression of apo(a) gene on growth of VSMCs and endothelial cells. Incubation of cultured medium of HepG2 cells transfected with apo(a) gene with human VSMCs or endothelial cells resulted in a significant increase in cell number compared with the conditioned medium of HepG2 cells transfected with control vector. In contrast, transfection of apo(a) gene directly into VSMCs caused no significant effect on VSMC growth. Therefore, we measured TGF-beta concentration in the conditioned medium of VSMCs. However, using ELISA, only latent but not active TGF-beta was detected in the medium of VSMCs. Moreover, addition of neutralizing anti-TGF-beta antibody did not alter VSMC growth. These results suggest that Lp(a) could stimulate growth of VSMCs via the independent mechanisms from the inhibition of TGF-beta activation. Overall, these data demonstrate that overexpression of apo(a) gene in cells producing apoB results in formation of Lp(a), resulting in a mitogenic action on human endothelial cells and VSMCs. These results provide new information to understand the mechanisms of the mitogenic action of Lp(a) and suggest the role of Lp(a) in the pathogenesis of atherosclerosis.

Regulatory effects of aggregated LDL on apoptosis during foam cell formation of human peripheral blood monocytes.

In order to investigate the mechanisms how modified lipoproteins enhance foam cell formation, we cultured peripheral blood monocytes with various stimulants and examined the effects of aggregated low-density lipoprotein (agLDL) on cell viability and lipid metabolism. AgLDL could completely inhibit phorbol ester-induced apoptosis, which was accompanied by intracellular cholesterol accumulation. Suppression of apoptosis-promoting proteases, ICE and CPP32, was observed in agLDL-treated cells. This indicates that agLDL accelerates foam cell formation through inhibition of apoptosis and enhancement of lipid accumulation in activated monocytes. By contrast, apoptosis was enhanced when monocytes were cultured with agLDL and M-CSF. Intracellular cholesterol accumulation was not significant in M-CSF treated cells. This suggests that M-CSF may act anti-atherogenic through apoptotic elimination of lipid-baring macrophages and enhanced lipid turnover. Our observation supports the novel hypothesis that regulation of apoptosis may play an important role in the development of atherosclerosis.

Quantitative immunoassay for IgA class circulating immune complexes using solid phase Facb fragment of anti-C3.

A quantitative assay of IgA class circulating immune complexes (IgA-CIC) by a solid phase anti-C3 enzyme immunoassay (anti-C3 EIA) is described. A stable and reproducible standard for determination of IgA-CIC was prepared successfully by chemical binding of complement C3 to human serum IgA. Two of 27 sera from patients with systemic lupus erythematosus (SLE), however, contained high concentrations of IgA class anti-F(ab')2 antibodies that caused false positive results when the F(ab')2 of anti-C3 was used for EIA. Solid phase Facb of anti-C3 was found to eliminate the false positive results caused by IgA class anti-F(ab')2 and IgA class rheumatoid factor. Good reproducibility and recovery were observed with this Facb anti-C3 EIA using the IgA-C3, a stable standard material, and so this method should be useful clinically in elucidating the role of IgA-CIC.

A human IgG1-K type M-protein with antibody activity against horse and rabbit alpha 2-macroglobulin.

A human IgG1-K type M-protein which specifically precipitates with alpha 2-macroglobulin (alpha 2-M) from horse and rabbit is described. This protein did not combine with human alpha 2-M nor with alpha 2-M from various other species. The binding activity was exclusively located in the Fab region of the molecule. This patient had never received injection of horse anti-tetanus serum. The present case seems to be the second report of an M-protein with antibody activity against horse and rabbit alpha 2-M.

Macro lactate dehydrogenase: an LDH-immunoglobulin M complex that inhibits lactate dehydrogenase activity in a patient's serum.

A macro lactate dehydrogenase (LDH, EC 1.1.1.27) isoenzyme with a low total LDH activity was present in the serum of a 57-year-old woman with a drug eruption (cutaneous lesions from an allergic reaction to drug administration). The patient's LDH was shown by immunoelectrophoresis to be bound to immunoglobulin G (IgG) and M (IgM). It was found that the patient's IgM acted as an inhibitor of LDH that was specific for the M subunit. When IgM was treated with 0.1 mol/l of 2-mercaptoethanol (2-ME), the inhibiting effect of the IgM to LDH activity disappeared, while treated IgM continued to bind to the LDH molecule. The LDH activity increased approximately two-fold when the patient's serum was treated with 2-ME. LDH activity in normal human serum was inhibited and an abnormal pattern of LDH isoenzyme appeared when the patient's IgM was added to normal serum. The present case seems to be the first report of LDH-IgM complex with a marked decrease of LDH activity.

Reference intervals for serum apolipoproteins A-I, A-II, B, C-II, C-III, and E in healthy Japanese determined with a commercial immunoturbidimetric assay and effects of sex, age, smoking, drinking, and Lp(a) level.

BACKGROUND: Apolipoproteins, which are contained in lipoprotein particles, play important roles in the transport of lipids. METHODS: Serum levels of apolipoproteins (apo) A-I, A-II, B, C-II, C-III, and E were determined by immunoturbidimetry in a healthy Japanese study population (1018 men and 1167 women, age 20-69 years) to establish reference intervals. RESULTS: Among the 2185 subjects examined, the mean serum value for apoA-I was 1.42 +/- 0.20 g/l, for apoA-II was 0.30 +/- 0.05 g/l, for apoB was 0.87 +/- 0.18 g/l, for apoC-II was 29 +/- 13 mg/l, for apoC-III was 75 +/- 20 mg/l, and for apoE was 36 +/- 9 mg/l. A sex difference was detected in the mean serum concentrations of all six apolipoproteins. Alcohol consumption and cigarette use had a slight effect on serum apolipoprotein concentrations. Age effects were observed among women in apoB, apoC-II, and apoC-III concentrations. Moreover, individuals with elevated serum lipoprotein (a) [Lp(a), >300 mg/l] also displayed increased serum apoB and apoC-II levels and an increased apoB/apoA-I ratio. CONCLUSION: The reference intervals for apolipoproteins in Japanese adults that we established, using commercially available reagents for automated analyzers, will be helpful for assessing risk of coronary heart disease and pathological conditions of patients with hyperlipidemia. We recommend use of these reference intervals for the clinical interpretation of serum apolipoprotein concentrations.

Pseudoleukocytosis without pseudothrombocytopenia induced by the interaction of EDTA and IgG(2)-kappa M-protein.

We encountered a patient who showed ethylenediaminetetraacetic acid (EDTA)-induced pseudoleukocytosis without pseudothrombocytopenia. The patient had IgG-kappa type monoclonal (M) gammopathy. The total protein concentration was 77 g/l, and the gamma-globulin fraction containing M-protein was 23.2%. The white blood cell count of the patient's blood anti-coagulated with EDTA was 52300/microl as determined using an automated counter, but was within normal limits when counted manually by light microscopy using a hemacytometer. Large amounts of a transparent substance were observed on blood smears, and white precipitates were formed by an interaction of the patient's serum with EDTA. Immunofixation electrophoresis showed these precipitates to be of the IgG(2)-kappa type M-protein. Western blotting analysis showed that the IgG molecules had a molecular mass of 155 kDa and were composed of two gamma-chains of approximately 53 kDa and two kappa-chains of 27 kDa. Pseudoleukocytosis was also observed when the patient's blood was anti-coagulated with O, O'-bis(2-amino-ethyl)-ethyleneglycol-N,N,N',N'-tetraacetic acid (EGTA) or sodium citrate, but not with lithium heparin. The present case seems to be the first report of pseudoleukocytosis induced by the interaction of EDTA and IgG(2)-kappa type M-protein.

A new Lp(a) assay that is unaffected by apo(a) size polymorphism.

We developed sandwich ELISA methods in which anti-apo(a) kringle 4 type 5 through protease (K4 x 5-Pro) domain monoclonal antibody (clone: 203E2) is employed in each instance as the capture antibody and one of the three species of monoclonal antibody [Mab] (clones: 108B8, 202A9, 2B3) is used as the labeled antibody. Using serum containing apo(a) with 34 repeats of kringle 4 as the calibrator, a commercial kit using anti-Lp(a) polyclonal antibody (Pab) or anti-apo(a) Mab overestimated the Lp(a) concentration in samples containing apo(a) with more than 34 repeats of kringle 4 and underestimated the Lp(a) concentration in samples containing apo(a) with fewer than 34 repeats of kringle 4. Moreover, it was demonstrated that the ratios of commercial kit values to anti-apo(a) K4 x 5-Pro Mab-based method values increased as the size of apo(a) increased. The ratios of apo(a) K5 x Pro Mab-based method values to anti-apo(a) K4 x 5-Pro Mab-based method values, however, remained almost constant regardless of the size polymorphism. Thus, we suggest that apo(a) size heterogeneity can significantly affect Lp(a) measurement in the Lp(a) assay using anti-Lp(a) Pab. The novel Lp(a) assay method, using only anti-apo(a) K4 x 5-Pro Mab, is not subject to this phenomenon.

Occurrence of heavy chain of 7S IgM half-molecule whose NH2-terminal sequence is identical with that of kappa light chain sequence in patients with Waldenstrom macroglobulinemia.

We report a rare case of a half molecule 7S IgM (HM 7SIgM) consisting of a unique mu heavy chain and kappa light chain found in blood and urine samples from a patient with primary Waldenstrom macroglobulinemia. A 64kDa abnormal immunoglobulin was detected in serum and urine by immunoblot method, and purified by a two-dimensional SDS-PAGE after separation from IgG and albumin fractions on gel filtration. NH2-terminal amino acid sequence analysis of the heavy chain revealed that residues 1-20 were identical to those of the NH2-terminal region of kappa light chain derived from the same patient. This sequence was then followed by a sequence that could not be identified by a computer homology search on the protein database. Using polypeptide segments obtained from the unique mu chain by digestion with endopeptidase, we identified a sequence spanning from residue 127 in the variable region of the known mu chain to residue 19 in the known CH1 domain and a sequence spanning from residues 67-82 in the heavy chain variable region class II. From these results, we concluded that the 64 kDa protein was an abnormal half molecule 7S IgM consisting of a kappa light chain and a unique mu heavy chain of 35 kDa polypeptide in which the NH2-terminal 20 amino acids were replaced by 20 amino acids derived from the sequence of kappa light chain in the NH2-terminal region.

Clinical significance of the serum lipoprotein(a) level in patients with systemic lupus erythematosus: its elevation during disease flare.

OBJECTIVE: To determine the clinical significance of serum lipoprotein(a) [Lp(a)] levels in patients with systemic lupus erythematosus (SLE). METHODS: Serum Lp(a) levels in 77 patients with SLE were measured by turbidimetric immunoassay. RESULTS: The median serum Lp(a) levels in all the SLE patients (14.4 mg/dl) and in those with active disease (Group A; 19.6 mg/dl, n = 39) at admission were significantly higher than those in healthy subjects (11.9 mg/dl, p < 0.05 and p < 0.001, respectively). The serum Lp(a) levels in SLE patients correlated directly with the serum cholesterol (p < 0.001) and urinary protein (p < 0.001) levels and inversely with the serum albumin levels (p < 0.02). Analysis limited to Group A patients with renal disease (Group A + RD, n = 28) revealed that the median serum Lp(a) level at the time of admission (OM) was significantly higher than those at 6 months before (-6M, p < 0.01) and at 6 months after admission (+6M, p < 0.01). Moreover, the serum Lp(a) level decrease from 0M to +6M in Group A+RD correlated significantly with the serum albumin level increase (p < 0.05). Multiple regression analysis demonstrated that the serum albumin level increment, the SLEDAI score decrement, the cholesterol level at 0M and the total dose of oral corticosteroids administered during the 0M to +6M period contributed independently and significantly to the serum Lp(a) level decrement from 0M to +6M in Group A + RD. CONCLUSION: Our study is the first to reveal that hypoalbuminemia appearing during disease flare plays an important role in increasing the serum Lp(a) levels in lupus patients with renal disease and shows that corticosteroid treatment reduced the elevated serum Lp(a) levels almost to original levels.

Serum lipoprotein lipase in healthy subjects: effects of gender and age, and relationships to lipid parameters.

Using serum samples obtained from normal individuals who had not received heparin injection, we investigated the relationship of lipoprotein lipase (LPL) to lipid and apolipoprotein concentrations in serum. For the measurement of LPL concentration, a highly sensitive enzyme-linked immunosorbent assay (ELISA) was developed, in which the lowest detection limit was 9 micrograms/L. The mean (SD) serum LPL concentration was 50.7 (14.9) micrograms/L (n = 240). It was lower in men [45.5 (14.0) micrograms/L in men and 56.0 (13.9) micrograms/L in women]. Serum LPL concentration correlated positively with high-density lipoprotein cholesterol (HDL-C, r = 0.549) and apolipoprotein AI (apoAI, r = 0.487), and inversely with triglycerides (r = -0.423). Our results suggest that serum LPL may be a factor in the modulation of HDL-C and triglyceride concentrations in normal subjects.

Reference values of plasma lipoprotein (a) in newly classified apolipoprotein(a) phenotype groups in the Japanese population.

We tried to establish the reference values of plasma lipoprotein (a) [Lp(a)] concentration in phenotype groups of apoliprotein(a) [apo A] classified by a new criterion. Lp(a) concentration was determined by latex agglutination immunoassay, and apo A was analysed by electrophoresis in sodium dodecyl sulphate-polyacrylamide gel and a Western blotting technique. According to the relative mobility to the apo B-100 band, apo A was classified into 11 isoforms, i.e. F, B, and S1-S9, and the phenotype was defined by their apparent combination. The frequency ratio of single-band versus double-band was approximately 2:1. In 382 cases of single-band, the most frequent phenotype was S5 (24.3%), followed by S4 (17.3%), S6 (15.4%) and S3 (14.4%). In 181 cases of double-band, S5/S6 phenotype was observed most frequently (12.2%). followed by S4/S5 (10.5%) and S3/S6 (7.2%). The reference value was determined between antilogs of the mean +/- 1.96 standard deviation by logarithmic transformation of all observed values for individual phenotype cases. These results suggest that the reference values shown to be variable with apo A phenotypes should be useful for evaluating Lp(a) values in diagnosis of atherosclerosis.

Determination of D-sorbitol in human erythrocytes by an enzymatic fluorometric method with an improved deproteinization procedure.

We developed an improved enzymatic assay of D-sorbitol in human erythrocytes by employing highly specific D-sorbitol dehydrogenase from Pseudomonas sp. (EC 1.1.1.14) and replacing perchloric acid (HClO4) and potassium carbonate (K2CO3), generally used for deproteinization, with sodium hydroxide (NaOH) and zinc sulphate (ZnSO4). In this assay, erythrocytes were separated from plasma by centrifugation and washed once with physiological saline. Subsequently, the erythrocytes were lysed with distilled water and proteins precipitated with NaOH and ZnSO4. After centrifugation, the resulting colourless supernatant was mixed with a glycine buffer (pH 9.0) containing NAD+ and D-sorbitol dehydrogenase. After incubation for 30 min at 37 degrees C, the NADH produced was measured fluorimetrically. The fluorescence intensities were corrected for sample blanks, and the values of D-sorbitol were normalized for haemoglobin content. The method had an analytical range of 1-180 micromol/L. The intra- and inter-assay precisions were < 3.3% and < 5.8%, respectively. The detection limit was 0.65 micromol/L. In terms of the linearity, precision and sensitivity, the improved method using NaOH and ZnSO4 was superior to the conventional method using HClO4 and K2CO3.

Identification and quantification of half-molecule immunoglobulins.

Abnormal IgA1 half-molecules consisting of one heavy and one light chain were found in a patient (NN) with typical multiple myeloma. Serum total protein was 9.4 g/dl, and the serum protein electrophoresis showed a monoclonal gamma-1 fraction amounting to 43.1%. The patient's serum and urine showed immunoelectrophoretically double arcs against anti-alpha antiserum having the same mobility. No double precipitin ring was demonstrated on single radial immunodiffusion analysis of the serum. The serum and the urine of this patient contained both 7.0S and 3.9S IgA-lambda type myeloma proteins. The IgA half-molecules (3.9S) were found to have a molecular weight of 59,000 daltons and were composed of one alpha-1 chain of about 40,000 daltons and one light chain of 22,000 daltons. Furthermore, enzymatic degradation suggested that the alpha chain of the half-molecules had a large deletion in its Fc portion. We suggest that its heavy and light chains were probably bound noncovalently, since the interchains connecting the heavy and light chains of these IgA half-molecules were easily dissociated with 1% SDS and 8M urea. The similar half-molecules of IgG type were also found in another patient of multiple myeloma.

[A case of Candida albicans endocarditis with impaired lung function].

A 53-year-old male was admitted to the hospital because of Candida albicans endocarditis. He had had a thoracoplasty due to pulmonary tuberculosis and showed severe restructive lung function. In 1987 and '89, trachiostomy was made because of respiratory failure. The patient was well until nine months earlier, when he consulted a physician because of fever. The investigations failed in finding the cause of the fever. He was administered antituberculosis agents and antiinflammatory drugs but had a fever every day. Two months before admission, a cardiac ultrasonographic study showed evident vegetations with mitral regurgitation. From the above course and examinations, a diagnosis of Candida albicans endocarditis was made. Infusions of CEZ, TOB, PIPC and miconazole for more than one month was ineffective. In November, 1990, he was referred to our medical center for the purpose of operation. A blood culture proved Candida albicans infection. An intravenous administration of fluconazole 400 mg/day was begun. However, there was pulmonary bleeding probably due to heparin used for prevention of atrial thrombosis and he developed fever, hypoexemia, ventricular tachycardia, and hyponatremia. He underwent mitral-valve replacement with a SJM valve. Culture of the vegetated mitral valve again proved Candida albicans. After operation, hypoexemia, ventricular tachycardia, hyponatremia were improved gradually. However he had an eosinophilia, eruption, and dyspnea. We suspected a drug eruption of fluconazole. Lymphocyte stimulating test of fluconazole proved positive. After the episode, he had no symptoms and was discharged.

[Coding for clinical laboratory information].

The field of clinical laboratory tests is facing an increase in the number of test items as well as a corresponding diversification due to the demands of medical institutions as well as improvements in analytical techniques. To respond to this situation, medical institutions have been promoting systematization of their testing procedures; information exchange among the institutions has likewise expanded with the use of media such as on-line systems and internet. Standardization of interfaces has been proposed to secure a common framework compatible with different types of information. Some embodiments in this country includes; (1) Interface Standards on Clinical Laboratory Information For information exchange, the format and reporting comments used in the media systems were standardized under the sponsorship of The Medical Information System Development Center, with a publication issued on 1993. (2) Standardization of Laboratory Test Code Standardization of codes for information exchange has been established under the sponsorship of The Japan Society of Clinical Pathology (Laboratory Test Coding Committee), through the systematization of laboratory test code used in media systems. A publication entitled "Classification & Coding for Clinical Laboratory Tests (8th edition in 1992, 9th edition in 1994 and supplement in 1996)" has been issued. The system for "Classification & Coding for Clinical Laboratory Tests" is divided into 5 components; (1) analyte code, (2) identification code, (3) specimen code, (4) methodology code, and (5) data classification code. The Laboratory test codes are precisely classified by "(1) analyte code", and then are identified by combination of additional codes such as specimen and methodology codes. In this year, we are making a new easily-used-codes composed of 5 Arabic figures.

[The clinical pathology for the twenty-first century].

At first, the ideal way of the scientific convention should be reexamined. It is approved the future direction which should do the joint with the related societies. It has been decided that it is held at a partly congruence with Japan Society of Clinical Chemistry in the next fiscal year. The Corporation Promotion Committee(chairman of prof. I. Sakurabayashi) negotiates with the Ministry of Education about the incorporation. The Society Improved Committee(chairman of prof. K. Watanabe) is discussing about a retirement system and improved select system of the councilor. And, though the more than 400 persons of clinical laboratory physicians has been registered as a certified clinical laboratory physicians, it copes in the selection committee of each university does not always taking laboratory medical doctor as a professor of the department of clinical laboratory. And, it becomes the name of the Japan Society of Clinical Pathology does not suit at present state. The Appellation Revision Subcommittee(chairman of K. Nakahara) is discussing in the ideal name of the Society. The opinion of the most part of way will concern national medical insurance. The clinical laboratory tests related groups(Japan Society of Clinical Pathology, Japanese Association of Clinical Laboratory Physicians, Japan Society of Medical technologists, Japan Registered Clinical Laboratories Association, Japan Association of Clinical Reagents Industries, Japan Council of Clinical Reagents wholesales) formed the Council on Clinical Laboratory Tests-Related Organization at present, and the demanding paper was submitted to related associations, such as Ministry of Health and Welfare, Japan Medical Association and so on.

[Diagnosis of HIV by clinical pathological methods].

HIV (human immunodeficiency virus)-I infection can be detected by the presence of HIV antigen, antibody to HIV, or gene of HIV itself in patient's blood. There are a considerable number of such antibody detecting methods (first generation) commercially available, but even the first was introduced in only mid-1985. These tests detect antibody in more than 90% of patients with confirmed AIDS. Recently, new antibody detecting methods (second generation) was produced using a single antigen (e.g. p 24, gp 41) of HIV-I by recombinant DNA or synthetic peptide. Furthermore, detection methods for DNA or RNA of HIV were developed and these methods are introduced in clinical use in near future.

[Effects on M-protein on laboratory data].

We describe two patients with abnormal immunoglobulins: Case 1 was a non-diabetic patient with IgA-kappa type M-protein whose serum fructosamine(FRA) value was markedly elevated; and Case 2 was a patient with pseudo-leukocytosis induced by the interaction of etylenediaminetetraacetic acid(EDTA) and IgG-kappa type M-protein. The M-protein in Case 1 was found to be conjugated to serum albumin by immunoelectrophoresis. By gel filtration, the FRA peak of the patient's serum was shown in the high molecule weight fraction. The glycation of IgA-kappa type M-protein was clearly demonstrated by FRA staining after fractionation by serum protein electrophoresis. Although serum FRA values of other non-diabetic patients with IgA type M-protein were elevated, patients with IgG type M-protein and IgM type M-protein had low or normal serum FRA values. In Case 2, the white blood cell count of the patient's blood anti-coagulated with EDTA was 52,300/microliter as determined using an automated counter, but was within normal limits when counted manually by light microscopy using a hemacytometer. The white precipitates were formed by the interaction of the patient's serum with EDTA. Immunofixation electrophoresis revealed that the precipitates were IgG2-kappa type M-protein.