Recommendations for the Use of Tryptase in the Diagnosis of Anaphylaxis and Clonal Mastcell Disorders.

Summary: Tryptase is a serin-protease produced and released by mast cells after IgE-mediated or non-IgE mediated stimuli. We here review the various aspects related to the molecular characteristics of the enzyme and its biological effects, the genetic basis of its production and the release kinetics. Recommendations for the clinical use of tryptase measurement developed by a task force of Società Italiana di Patologia Clinica e Medicina di Laboratorio and Associazione Allergologi Immunologi Italiani Territoriali e Ospedalieri are given on the best procedure for a correct definition of the reference values in relation to the inter-individual variability and to the correct determination of tryptase in blood and other biological liquids, in the diagnosis of anaphylaxis (from drugs, food, insect sting, or idiophatic), death from anaphylaxis (post mortem assessment) and cutaneous or clonal mastcell disorders.

Combining immunofluorescence with immunoblot assay improves the specificity of autoantibody testing for myositis.

OBJECTIVE: Immunoblot (IB) methods are widely used to detect myositis-specific autoantibodies (MSAs); however, false-positive results are common. In this study, we aimed to determine whether associating the anti-nuclear antibody (ANA) IIF pattern may help to improve the specificity of MSA detection by IB in patients with idiopathic inflammatory myositis (IIM). METHODS: Serum samples from 104 patients presenting with muscle weakness/myalgia and positive to at least one MSA by IB (MYOS12 Diver and MIOS7 Diver, D-tek) were tested for ANAs on HEp-2000 cells (Immuno Concepts). The chi-square test was used to analyse the concordance of the MSA result and its corresponding pattern by ANA testing between patients with and without IIM. RESULTS: Eighty-three of the 104 patients had a diagnosis of definite IIM, while in 21 cases, patients were affected by other autoimmune diseases or various non-systemic diseases. Forty nine of 83 (59%) patients in the IIM group and 4/21 (19%) in the non-IIM group showed a concordance between ANA pattern and MSAs by IB (P < 0.001). MSA monopositivity was significantly associated with IIM (91.6%) compared with 61.9% in the non-IIM group (P = 0.0005). CONCLUSIONS: Considering both the MSA result and its corresponding pattern by ANA testing may help to improve the specificity of MSA detection by IB and to confirm the diagnosis of MSA-associated IIM. The monopositivity of MSAs is an important additional tool to validate IB results.

Recommendations for the use of molecular diagnostics in the diagnosis of allergic diseases.

Summary: The Study Group on Allergology of the Italian Society of Clinical Pathology and Laboratory Medicine (SIPMeL) and the Associazione Italiana degli Allergologi e Immunologi Territoriali e Ospedalieri (AAIITO) developed the present recommendations on the diagnosis of allergic diseases based on the use of molecular allergenic components, whose purpose is to provide the pathologists and the clinicians with information and algorithms enabling a proper use of this second-level diagnostics. Molecular diagnostics allows definition of the exact sensitization profile of the allergic patient. The methodology followed to develop these recommendations included an initial phase of discussion between all the components to integrate the knowledge derived from scientific evidence, a revision of the recommendations made by Italian and foreign experts, and the subsequent production of this document to be disseminated to all those who deal with allergy diagnostics.

Mutations in MYH9 result in the May-Hegglin anomaly, and Fechtner and Sebastian syndromes. The May-Heggllin/Fechtner Syndrome Consortium.

The autosomal dominant, giant-platelet disorders, May-Hegglin anomaly (MHA; MIM 155100), Fechtner syndrome (FTNS; MIM 153640) and Sebastian syndrome (SBS), share the triad of thrombocytopenia, large platelets and characteristic leukocyte inclusions ('Döhle-like' bodies). MHA and SBS can be differentiated by subtle ultrastructural leukocyte inclusion features, whereas FTNS is distinguished by the additional Alport-like clinical features of sensorineural deafness, cataracts and nephritis. The similarities between these platelet disorders and our recent refinement of the MHA (ref. 6) and FTNS (ref. 7) disease loci to an overlapping region of 480 kb on chromosome 22 suggested that all three disorders are allelic. Among the identified candidate genes is the gene encoding nonmuscle myosin heavy chain 9 (MYH9; refs 8-10), which is expressed in platelets and upregulated during granulocyte differentiation. We identified six MYH9 mutations (one nonsense and five missense) in seven unrelated probands from MHA, SBS and FTNS families. On the basis of molecular modelling, the two mutations affecting the myosin head were predicted to impose electrostatic and conformational changes, whereas the truncating mutation deleted the unique carboxy-terminal tailpiece. The remaining missense mutations, all affecting highly conserved coiled-coil domain positions, imparted destabilizing electrostatic and polar changes. Thus, our results suggest that mutations in MYH9 result in three megakaryocyte/platelet/leukocyte syndromes and are important in the pathogenesis of sensorineural deafness, cataracts and nephritis.

Diagnosing systemic lupus erythematosus: new-generation immunoassays for measurement of anti-dsDNA antibodies are an effective alternative to the Farr technique and the Crithidia luciliae immunofluorescence test.

The aim of this study was to evaluate the diagnostic performance of four new enzyme immunoassays (EIAs) for anti-double-stranded-DNA (anti-dsDNA) antibodies, in comparison with the Farr assay and the Crithidia luciliae immunofluorescence test (CLIFT). To this purpose, sera from four patient groups were collected: 52 sera from patients with systemic lupus erythematosus (SLE); 28 from patients with other connective tissue diseases (CTD); 36 from patients with hepatitis C virus (HCV) infection; and 24 from those with acute viral infection. All sera were tested for anti-dsDNA antibodies by four EIA methods using a different antigenic DNA source [synthetic oligonucleotide (Method A), circular plasmid (Method B), recombinant (Method C), and purified extracted (Method D)], and by CLIFT and Farr assays. The diagnostic sensitivity of the assays was as follows: 84.6% (Method A), 73% (B), 82.7% (C), 84.6% (D), 55.8% (CLIFT), and 78.8% (Farr). Specificity was 82.9% (A), 97.7% (B), 96.5% (C), 94.3% (D), 96.5% (CLIFT), and 90.9% (Farr). From these data, we can conclude that the new-generation EIA methods evaluated in this study have higher sensitivity than the CLIFT and Farr assays and, with the exception of Method A, have specificity similar to the CLIFT and slightly higher than the Farr assay. These findings suggest that EIA tests may replace CLIFT as a screening test and the Farr assay as a specific test, for anti-dsDNA antibody detection.

Diagnostic accuracy of different immunological methods for the detection of antineuronal antibodies in paraneoplastic neurological syndromes.

The aim of this work was to evaluate the diagnostic accuracy of three different analytical methods for the detection of antineuronal antibodies and outline how they might be used to diagnose Paraneoplastic Neurological Syndromes (PNS) in a more effectively and rationally way. One hundred and four patients with neurological diseases were studied: 38 with paraneoplastic neurological disorder, 44 with other neurological diseases, and 22 with systemic autoimmune diseases and neurological disorders. 20 healthy subjects and 18 subjects with tumour without neurological disorders were also studied. Antineuronal antibodies were tested using three methods: Western blot (WB); Line-blot (LB); and indirect immunofluorescence (IIF) on primate cerebellum. The diagnostic sensitivity of the IIF, WB and LB methods was 28.9%, 26.3% and 36.8%, respectively, and their specificity was 95.2%, 97.1% and 98.1% respectively. The combined use of the three methods brought the sensitivity to 39.4%. The results of this study show that the methods used in clinical laboratories for the detection of antineuronal antibodies have good specificity. Among the three methods assessed, LB showed the highest diagnostic accuracy and also allowed for recognition of fine antibody specificities. According to these results we can suggest that LB should be used as the method of choice to search for paraneoplastic antibodies.

Detection of a novel 20 kDa shrimp allergen showing cross-reactivity to house dust mites.

BACKGROUND: Allergy to crustacean shellfish is one of the most common IgE-mediated food allergies, and tropomyosin has been identified as the major allergen. However, not all subjects affected by this allergy are IgE-positive to tropomyosin. AIMS: To evaluate whether sera of patients with shrimp allergy but negative for tropomyosin react to other allergen(s); and to evaluate the role such allergen(s) may play in cross-reactivity between crustaceans and house dust mites (HDMs). METHODS: Three different pools of sera-one from subjects with shellfish allergy and HDMs positivity, but negative for recombinant and native tropomyosin (rPen a 1 and nPen m 1) (Pool 2); a second from subjects with tropomyosin and HDMs positivity (Pool 1); and the last from subjects allergic only to HDMs (Pool 3) were submitted to immunoblotting. Subsequently, a 20 kDa protein- enriched fraction of shrimp extract was used at two different concentrations (10 and 100 microg/mL) to pre-absorb the Pool 2 serum and to evaluate, by ELISA assay, the level of inhibition on shrimp and HDMs-coated wells, respectively. RESULTS: The Pool 2 serum showed IgE reactivity against a 20 kDa component. Its pre-absorption with an enriched fraction of 20 kDa protein caused an inhibition of 56% in IgE binding to shrimp extract at a concentration of 100 microg/mL, and of 14% and 35% to HDMs extract at concentrations of 10 and 100 microg/mL, respectively, as measured by ELISA assay. CONCLUSIONS: The 20 kDa component seems to be a new crustacean allergen and it could play a role in cross-reactivity with HDMs.

[Accuracy and standardization of diagnostic methods for the detection of antibodies to citrullinated peptides]. / Accuratezza diagnostica e standardizzazione dei metodi per la determinazione degli anticorpi anti-peptidi citrullinati.

Anti-citrullinated peptide antibodies (ACPA) have a very high specificity for rheumatoid arthritis, much more than that of the rheumatoid factor. In addition, ACPA can be found in sera in the pre-clinical phase, are associated with more severe joint destruction and with higher disease activity. In recent years, keeping pace with new knowledge and with progress made in the antigenic composition of tests and in the characterization of immunogenic epitopes, many immunoenzymatic (ELISA) methods of second and third generation have been produced and marketed commercially, and their use has spread among clinical laboratories. Today, completely automated methods are also available, which are easy to use and with a higher throughput, rendering the diagnostic utility of testing ever faster and more effective. This review takes into consideration the more important characteristics of the new ACPA-ELISA tests now commercially available, and also considers recent progress in standardizing test results.

Anti-prothrombin antibodies predict thrombosis in patients with systemic lupus erythematosus: a 15-year longitudinal study.

OBJECTIVE: To evaluate the role of anti-prothrombin (anti-PT) antibodies in predicting thrombosis in patients with systemic lupus erythematosus (SLE). METHODS: An inception cohort of 101 SLE patients (12 males, 89 females; mean age 30 +/- 8 years), was considered. Clinical and laboratory evaluations were regularly performed during a 15-year follow-up (median 108 months) with a special focus on thromboembolic events. Serum samples were collected at time of diagnosis and at least once a year thereafter. IgG and IgM anti-PT, anti-cardiolipin (aCL) and anti-beta(2)glycoprotein I (beta(2)GPI) antibodies were measured by enzyme-linked immunosorbent assay (ELISA); lupus anticoagulant (LAC) was assayed by the dilute Russell's viper venom time and activated partial thromboplastin time tests. The analytical specificity of anti-PT ELISA was investigated. The timing of thrombosis occurrence was calculated using the Kaplan-Meier method. RESULTS: In the 15-year follow-up, thrombosis occurred in 14 out of the 101 patients: venous thrombosis in nine cases and arterial thrombosis in five. IgG and/or IgM anti-PT, anti-beta(2)GPI and aCL antibodies, and LAC activity were detected in ten, nine, seven, and nine cases, with sensitivity for thrombosis of 71.4%, 64.3%, 50% and 64.3%, respectively. Thrombosis-free survival was 90% at 5 years and 85.8% at 10 and 15 years, respectively. Thrombosis was predicted by anti-PT (P = 0.001), anti-beta(2)GPI antibodies (P = 0.002) and LAC activity (P = 0.001). Moreover, the risk of thrombosis progressively increased with the number of positive antiphospholipid antibody tests. The presence of four positive antibody tests was associated with a risk of thrombosis thirtyfold higher than in their absence. CONCLUSIONS: This longitudinal study shows that IgG anti-PT antibodies are predictors of thrombosis in SLE patients.

Diagnostic accuracy of IgA anti-tissue transglutaminase antibody assays in celiac disease patients with selective IgA deficiency.

Clinical studies have estimated a 10- to 20-fold increased risk for celiac disease (CD) in patients with selective IgA deficiency (SIgAD). For this reason, screening for CD is mandatory in SIgAD patients, but it represents a special challenge since the specific IgA class antibodies against gliadin (AGA), endomysium (EMA), and tissue-transglutaminase (tTG) are not produced in patients with CD. IgG class counterparts of these antibodies may be informative; in particular IgG EMA has been demonstrated to be a valid marker for diagnosing CD in SIgAD cases, but it is not used much in clinical laboratories, because it is cumbersome and involves some technical difficulties. Even if it was widely used in clinical laboratories, the measuring of IgG AGA has shown a less-than-optimum diagnostic accuracy, so that now it tends to be substituted by tests for anti-tTG IgG, for which the few available studies have shown diagnostic performances superior to AGA. Since it is not known whether various available methods for measuring IgG anti-tTG antibodies offer similar diagnostic performances, we have compared the results obtained from nine second-generation commercial methods (D-tek, Phadia, Immco, Orgentec, Radim, Euroimmun, Inova, Aesku, Generic Assays), measuring IgG anti-tTG antibodies in 20 patients with CD and SIgAD and in 113 controls (9 patients with SIgAD without CD, 54 patients with chronic liver disease, and 50 healthy individuals). Diagnostic sensitivity, calculated by means of ROC plot analysis, ranged between 75% and 95%, and specificity ranged from 94% to 100%. In the same population, the diagnostic sensitivity and specificity of AGA IgG were 40% and 87%, respectively. Even though they perform differently, all IgG anti-tTG methods evaluated are reliable serological assays for the diagnosis of CD in SIgAD patients, with diagnostic accuracy superior to the AGA IgG method. The methods that use a mix of tTG and gliadin peptides as the antigenic preparation have a specificity slightly lower than that of the methods that use only tTG.

Antiprothrombin antibodies: a comparative analysis of homemade and commercial methods. A collaborative study by the Forum Interdisciplinare per la Ricerca nelle Malattie Autoimmuni (FIRMA).

OBJECTIVE: Prothrombin (PT) is a target for antibodies with lupus anticoagulant (LA) activity, suggesting the possible application of anti-prothrombin antibody (aPT) assays in patients with antiphospholipid syndrome (APS). Different methods - both homemade and commercial - for the detection of aPT are available, but they seem to produce conflicting results. The purpose of this study was to compare the performance of different assays on a set of well-characterized serum samples. PATIENTS AND METHODS: Sera were gathered from 4 FIRMA institutions, and distributed to 15 participating centres. Forty-five samples were from patients positive for LA and/or anticardiolipin antibodies (aCL) with or without APS, and 15 were from rheumatoid arthritis (RA) patients negative for antiphospholipid antibodies. The samples were evaluated for IgG and IgM antibodies using a homemade direct aPT assay (method 1), a homemade phosphatidylserine-dependent aPT assay (aPS/PT, method 2), and two different commercial kits (methods 3 and 4). In addition, a commercial kit for the detection of IgG-A-M aPT (method 5) was used. RESULTS: Inter-laboratory results for the 5 methods were not always comparable when different methods were used. Good inter-assay concordance was found for IgG antibodies evaluated using methods 1, 3, and 4 (Cohen k > 0.4), while the IgM results were discordant between assays. In patients with thrombosis and pregnancy losses, method 5 performed better than the others. CONCLUSION: While aPT and aPS/PT assays could be of interest from a clinical perspective, their routine performance cannot yet be recommended because of problems connected with the reproducibility and interpretation of the results.

An effective algorithm for the serological diagnosis of idiopathic inflammatory myopathies: The key role of anti-Ro52 antibodies.

BACKGROUND: Patients with suspected idiopathic inflammatory myopathies (IIM) are commonly tested for the presence of anti-nuclear antibodies (ANA) by indirect immunofluorescence (IIF) on HEp-2 cell substrates. However, ANA-IIF false negative tests may occur in IIM because some antigens, such as Jo1 and Ro52, may be scarcely expressed on HEp-2 cells. In addition, cytoplasmic staining is often not appropriately investigated by a specific antibody assay, leading to decreased clinical sensitivity of the ANA test. We evaluated the diagnostic impact of different strategies using different combination of myositis-related autoantibody tests. METHODS: Sera from 51 patients with an established diagnosis of IIM were tested for ANA by IIF on HEp-2 cells and for myositis-specific antibodies (MSA) and myositis-associated antibodies (MAA) by lineblot methods. RESULTS: Forty-four/51 (86.3%) samples tested positive with at least one of the three methods and seven were negative with all methods. Of the 44 positive samples, 9 (20.5%) tested negative for the ANA-IIF test and positive for MAA/MSA. Anti-Ro52 were the most prevalent autoantibodies in IIM patients (21/51; 41%), frequently associated with anti-Jo1 antibodies (13/21; 62%). 13 (16%) anti-Ro52 and anti-Jo1 negative samples were reactive to MSA. CONCLUSIONS: Our findings suggest that when IIM is clinically suspected, the optimal diagnostic algorithm is to associate the ANA-IIF screening test with a specific test for anti-Ro52 and anti-Jo1 antibodies. Should all these tests be negative, serological tests for MSA are recommended.

Definition of the upper reference limit for thyroglobulin antibodies according to the National Academy of Clinical Biochemistry guidelines: comparison of eleven different automated methods.

PURPOSE: In the last two decades, thyroglobulin autoantibodies (TgAb) measurement has progressively switched from marker of thyroid autoimmunity to test associated with thyroglobulin (Tg) to verify the presence or absence of TgAb interference in the follow-up of patients with differentiated thyroid cancer. Of note, TgAb measurement is cumbersome: despite standardization against the International Reference Preparation MRC 65/93, several studies demonstrated high inter-method variability and wide variation in limits of detection and in reference intervals. Taking into account the above considerations, the main aim of the present study was the determination of TgAb upper reference limit (URL), according to the National Academy of Clinical Biochemistry guidelines, through the comparison of eleven commercial automated immunoassay platforms. METHODS: The sera of 120 healthy males, selected from a population survey in the province of Verona, Italy, were tested for TgAb concentration using eleven IMA applied on as many automated analyzers: AIA-2000 (AIA) and AIA-CL2400 (CL2), Tosoh Bioscience; Architect (ARC), Abbott Diagnostics; Advia Centaur XP (CEN) and Immulite 2000 XPi (IMM), Siemens Healthineers; Cobas 6000 (COB), Roche Diagnostics; Kryptor (KRY), Thermo Fisher Scientific BRAHMS, Liaison XL (LIA), Diasorin; Lumipulse G (LUM), Fujirebio; Maglumi 2000 Plus (MAG), Snibe and Phadia 250 (PHA), Phadia AB, Thermo Fisher Scientific. All assays were performed according to manufacturers' instructions in six different laboratories in Friuli-Venezia Giulia and Veneto regions of Italy [Lab 1 (AIA), Lab 2 (CL2), Lab 3 (ARC, COB and LUM), Lab 4 (CEN, IMM, KRY and MAG), Lab 5 (LIA) and Lab 6 (PHA)]. Since TgAb values were not normally distributed, the experimental URL (e-URL) was established at 97.5 percentile according to the non-parametric method. RESULTS: TgAb e-URLs showed a significant inter-method variability. Considering the same method, e-URL was much lower than that suggested by manufacturers (m-URL), except for ARC and MAG. Correlation and linear regression were unsatisfactory. Consequently, the agreement between methods was poor, with significant bias in Bland-Altman plot. CONCLUSIONS: Despite the efforts for harmonization, TgAb methods cannot be used interchangeably. Therefore, additional effort is required to improve analytical performance taking into consideration approved protocols and guidelines. Moreover, TgAb URL should be used with caution in the management of differentiated thyroid carcinoma patients since the presence and/or the degree of TgAb interference in Tg measurement has not yet been well defined.

A prospective study of 1038 pregnancies on the predictive value of anti-annexin V antibodies for fetal loss.

Retrospective studies have demonstrated that anti-annexin V (anti-AnxV) antibodies are linked to miscarriage. Their predictive value is, however, unknown. We have carried out a prospective study to evaluate the relationship between anti-AnxV antibodies and the pregnancy outcome. A serum sample was taken from 1038 consecutive healthy women at the beginning of pregnancy. IgG and IgM anti-AnxV antibodies were measured by an ELISA method. The cutoff value was set at 5 units for both IgG and IgM. Out of 1038 women, 116 (11.4%) had a miscarriage by the 22nd week; 10 were lost to follow-up, 10 had an induced abortion, 6 had a preterm delivery, and 896 carried their pregnancy through to term. An adverse outcome of the pregnancy proved to be directly related to the number of previous miscarriages (P = .008) and the age of the woman (P = .002). IgG and IgM anti-AnxV were present in 25% and 27% of the women who miscarried, and in 23% and 28% of those who gave birth (mean antibody concentration IgG, 4.2 vs. 4.4 U/mL; IgM, 3.7 vs. 3.5 U/mL). IgG and IgM anticardiolipin and anti-beta(2)GPI, together with antinuclear, antithyroperoxidase, and antithyroglobulin antibodies, were also measured in the 116 sera of the women with miscarriage and in an equal number of women who gave birth. Their positivity or level proved not to be useful in discriminating between the risk of miscarriage and term delivery. This large-scale prospective study demonstrates that the presence of IgG and IgM anti-AnxV antibodies, when measured in healthy women, does not give a positive predictive lead towards the possibility of a miscarriage, and it is not useful in evaluating the risk of miscarriage at the beginning of pregnancy.

Accuracy of semiquantitative immunoenzymatic methods in quantitation of anti-topoisomerase I (Scl-70) antibodies.

Reports of a possible correlation between anti-Scl-70 antibody concentration and clinical manifestations in systemic sclerosis patients have recently appeared in the scientific literature. The goal of our study was to evaluate, by means of a multicenter study, the analytical reliability of immunoassay systems in the quantitative measurement of Scl-70 antibodies. Three blind samples (H, M, L) at different anti-Scl-70 antibody concentrations, and a low concentration antibody serum (LPC) used as a common calibrator, were sent three times in a 6-month time span to 39 Italian clinical laboratories. Each laboratory was asked to calculate dosages following the enzyme-linked immunosorbent assay (ELISA) method they used and report the optical density values of each sample (ODs), of the cutoff serum provided by the manufacturer of the kit used (ODco) and of LPC (ODLPC). The overall analytical imprecision (between methods and between laboratories) of the three different determinations of the values respectively expressed in ODs, ODs/ODco and ODs/ODLPCratio was 47.1, 52.8 and 34.0% for sample H, 56.2, 47.4% and 34% for sample M and 84.6, 86.0 and 86.6% for sample L. The average intra-method analytical imprecision was, respectively, 20.7, 29.8 and 18.6% for sample H, 24.6, 26.5 and 19.3% for sample M, and 30.6, 28.1 and 20.2% for sample L. The commercial ELISA methods currently used to determine the presence of anti-Scl-70 autoantibodies show considerable differences in the quantitative determination. The best results for reproducibility analyses have been obtained when the values were expressed as a ratio between the ODs of the sample and of the common calibrator (ODs/ODLPC). Forward-looking clinical studies that can clarify the usefulness of quantitative determination of anti-Scl-70 antibodies in the monitoring of diffuse scleroderma patients can be performed only when standard serum with a known antibody concentration and calibration curves for quantitative ELISA measurements are made available.

Variability between methods to determine ANA, anti-dsDNA and anti-ENA autoantibodies: a collaborative study with the biomedical industry.

This study was performed by the Italian Society of Laboratory Medicine (SIMeL) in order to establish the variability between the different analytical systems currently used in clinical laboratories for the detection of autoantibodies diagnostic of systemic autoimmune disease. Sixteen industrial, and two university laboratories participated in this study which entailed the determination of anti-nuclear (ANA), anti-dsDNA and anti-ENA antibodies in 11 sera from patients with clinically diagnosed systemic rheumatic disease, using reagents produced by these companies and different methodologies (indirect immunofluorescence, immunoenzymatic assay, counterimmunolectrophoresis, immuno and western blotting). We found 93.5% agreement between the methods used for the detection of ANA, 85.2% for anti-dsDNA antibodies, and 86.9% for anti-ENA antibodies. Among the anti-ENA antibodies, regardless of the method used, detection percentages were excellent for anti-RNP and anti-SSB/La (100%), good for anti-SSA/Ro (93%), but unacceptable for the anti-Jo-1 (67%), anti-Scl70 and anti-Sm (47%) antibodies. This further stresses the need for rigorous standardisation of commercial reagents and analytical procedures, as well as the introduction of external quality assessment (EQA) programs, and a complete definition of operative protocols adjusted to the sensitivity and specificity of the various methods.

EDTA-dependent pseudothrombocytopenia. Association with antiplatelet and antiphospholipid antibodies.

In a study of 88 patients with EDTA-dependent pseudothrombocytopenia (PTCP), EDTA-dependent antiplatelet antibodies were seen in the sera of 72 (81.8%) patients (44 IgM, 25 IgG, and 3 IgA). The same sera also were tested for anticardiolipin antibodies (aCL), and 56 (63.6%) patients had sera that also were reactive for aCL (33 IgM, 21 IgG, and 2 IgA). The 16 patients who were negative for antiplatelet antibodies also were negative for aCL antibody. Overall concordance between antiplatelet and aCL antibodies was 82.9%; the correlation between antiplatelet and aCL antibody isotype distribution was 82.1%. Following cardiolipin absorption, most of the PTCP-sera were negative for antiplatelet activity, and no longer reproduced platelet clumping when incubated with normal blood. This finding showed that the antiplatelet antibodies cross-reacted with negatively charged phospholipids. However, after absorption on normal platelets, complete inhibition of aCL activity was observed in 34 (60.7%), and partial inhibition in 14 of the 56 patients who were aCL positive. These findings support the hypothesis that antibody subpopulations (naturally occurring autoantibodies) directed against negatively charged phospholipids can bind to antigens modified by EDTA on the platelet membrane, and may be responsible for PTCP genesis.

Eosinophilic peroxidase deficiency. Cytochemical and ultrastructural characterization of 21 new cases.

Morphologic, instrumental (flow cytometric), cytochemical, ultrastructural, and chromosomal studies were performed in 21 cases of eosinophilic peroxidase deficiency that were observed in an area of northeastern Italy in the last 5 years. It was found that eosinophilic peroxidase deficiency occurred with a frequency of 1 case in 14,000 complete blood counts yearly, and thus is less rare than previously thought. Eosinophils appeared morphologically normal when examined using the light microscope, but ultrastructural study disclosed several aspecific granule alterations. In the first family studied, members with partial and total deficit were identified; in all the other cases, the enzyme deficit was total (negative cytochemical reactions and absence of dimethylaminoazobenzene-positive specific granules at the electron microscope), isolated (a single affected member in each family examined), and stable (persistent at long-term follow-up). Eosinophilic peroxidase deficiency was not correlatable with any particular disease, although a nonsignificant association with allergic-type conditions was observed. Studies are in progress to examine the modality of the defect's genetic transmission, as well as problems related to possible functional alterations and correlated clinical consequences.

Platelet satellitism is Fc gamma RIII (CD16) receptor-mediated.

Platelet satellitism (PS), the phenomenon of platelet rosetting around polymorphonuclear neutrophils (PMN), which is observed in ethylenediamineetetraacetic acid (EDTA)-anticoagulated blood at room temperature, is caused by the presence of IgG autoantibodies in the serum. Fourteen patients with PS were studied, and the presence of both EDTA-dependent antiplatelet and EDTA-dependent antineutrophil IgG (auto)antibodies were found in their sera. Anti-neutrophil activity was completely abolished when the sera were absorbed on normal platelets, which suggests that a single antibody is involved. Inhibition studies with monoclonal antibodies indicated that this IgG autoantibody is directed against the glycoprotein IIb/IIIa complex of the platelet membrane, as well as the neutrophil Fc gamma receptor III (Fc gamma RIII). In addition, the antibody did not react with platelets from a patient with type I Glanzmann's disease, nor with neutrophils from a patient with congenital Fc gamma RIII absence (NAnull phenotype), thus confirming both specificities. As in other literature cases, a clear correlation between the presence of this IgG and a specific clinical situation, disease, or use of drugs could not be shown. Therefore, these antibodies, which are present in some normal individuals, might occur naturally. Because of the exposure of particular cryptoantigenic structures present on EDTA-modified platelet and PMNs, they may manifest themselves by triggering the PS phenomenon.

IgG platelet antibodies in EDTA-dependent pseudothrombocytopenia bind to platelet membrane glycoprotein IIb.

EDTA-dependent pseudothrombocytopenia (PTCP) consists of an inappropriate low platelet count caused by autoantibodies present in the serum samples reacting with platelets only in EDTA-anticoagulated blood. By using immunoprecipitation and Western blot techniques, we studied the immunochemical specificity of platelet agglutinating autoantibodies in the serum samples of 10 patients with PTCP. Furthermore, to evaluate a possible role of PTCP-associated IgG autoantibodies in increased platelet turnover, we assayed the plasma glycocalicin (GC) level and calculated the GC index for every patient. Our results provide direct evidence that an epitope located on platelet membrane glycoprotein IIb is recognized by PTCP-associated IgG antibodies; moreover GC levels in patients with EDTA-dependent PTCP were similar to control levels, thus excluding an increased platelet turnover. We conclude that antiplatelet antibodies directed against platelet cryptantigens are unlikely to have a major role in the increased removal of cells from circulation.