Validation of automated latex von Willebrand factor activity assay in a new prospective study including post-treatment patient samples.

INTRODUCTION: Von Willebrand disease (VWD) is the most prevalent inherited bleeding disorder. Diagnosis requires measurement of VWF-platelet binding function, for which VWF ristocetin cofactor activity (VWF:RCo) is the reference method. Recently, an automated latex particle-enhanced immunoturbidimetric von Willebrand factor activity assay (VWF:Ab) has been validated showing superior characteristics. We further validate VWF:Ab in a prospective study including post-treatment patient samples. METHODS: A total of 1151 samples were collected from patients tested for VWD, including 119 samples from patients treated with desmopressin or VWF replacement product. All samples were tested for VWF:Ab and VWF:RCo, and the methods were compared using linear regression. Imprecision, linearity and lower detection limit were determined for both assays. RESULTS: VWF:Ab showed improved precision compared to VWF:RCo. Linear regression of VWF:Ab and VWF:RCo across all samples exhibited good agreement (R2 = 0.89) with statistical significance (P < 0.001) and bias of -8.7. Concordance was high in classifying samples as normal or abnormal. Analysis of treated samples showed excellent agreement (R2 = 0.91) with statistical significance (P < 0.001) and bias of -4.3. CONCLUSIONS: Our analysis validates the VWF:Ab assay in a prospective study of a large cohort of patient samples and extends these results to post-treatment patient samples.

Implementación y evaluación de un programa de gestión de calidad para el monitoreo de glucómetros en un hospital universitario / Implementation and evaluation of a management program quality for monitoring glucometers in a hospital academic

Introducción: El uso de dispositivos portátiles para controlar la glucemia se ha extendido en los últimos años a las entidades hospitalarias, porque proporcionan un resultado rápido al realizarse al lado de la cama del paciente (point-of-care testing). Objetivo: Describir el proceso y los resultados de la implementación de un programa de gestión de calidad para el control de glucómetros hospitalarios. Materiales y Métodos: Se presenta la implementación de un programa de gestión de calidad para evaluar 50 glucómetros pertenecientes a siete áreas críticas del Hospital Italiano de Buenos Aires (Unidad Coronaria, Terapia Intensiva de Adultos y Pediátrica, Terapia Intermedia, Central de Emergencias de Adultos y Pediátrica, Unidad de Cuidados Intensivos Neonatológicos), desde el 1 de enero de 2014 hasta la actualidad, basado en tres estrategias: control diario, análisis mensual (precisión y exactitud) y control de muestras paralelas. Resultados: Luego de instaurar este programa y analizar los datos de los primeros 17 meses, se requirió un recambio total de 292 glucómetros: 150 debido al control de calidad diario, 119 por el análisis mensual y 23 por el control de muestras paralelas. Esto implicó retirar 17/50 glucómetros por mes. Conclusiones: Si bien estos dispositivos son útiles por su rápida respuesta, el 34,6% debió ser reemplazado por no haber superado alguno de los requisitos planteados en las estrategias de evaluación. La peor performance fue en los niveles de hipoglucemia, situación de interés para la rápida toma de decisiones. Es importante destacar la necesidad de aplicación de un plan de calidad para glucómetros sobre la base de un diseño propio y a medida de la institución para garantizar la seguridad del paciente.(AU)

Introduction: The use of portable devices for glycemic control has been extended in recent years to hospital entities, because they provide a rapid result when they are performed at or near the patient's bedside (point of care testing). Objective: To describe the process and results of the implementation of a quality management program to control of hospital glucometers. Materials and Methods: We present the implementation of a quality management program to evaluate 50 glucometers belonging to seven critical areas of Hospital Italiano de Buenos Aires (Coronary Unit, Adult and Pediatric Intensive Care Unit, Intermediate Therapy, Adult and Pediatric Emergency Center, Unit of Neonatal Intensive Care), from January 1, 2014 to the present, based on three strategies: Daily Control, Monthly Analysis and Control of Parallel Samples. Results: After implementing this program and analyzing the first 17 months, the substitution of 292 glucometers was required: 150 due to daily quality control, 119 per monthly analysis and 23 due to control parallel samples. This involved withdrawing 17/50 glucometers monthly. Conclusions: Although these devices are useful because of their rapid response, 34.6% had to be replaced because they exceeded the requirements presented in the evaluation strategies. The worst performance was in levels of hypoglycemia, a situation of interest for rapid decision-making. It is important to emphasize the need to apply a quality plan for glucometers based on an own design and suitable for the institution to guarantee the safety of the patient.(AU)

Centrifugation speed affects light transmission aggregometry.

BACKGROUND: Light transmission aggregometry (LTA) is considered the gold standard for investigating platelet activity ex vivo. However, LTA protocols are not standardized, and differences in LTA procedure are a potential source of variance in results. Centrifugation speed is an essential component of platelet preparation in LTA, has yet to be standardized, and may affect platelet aggregation results. We sought to investigate the effect of relative centrifugal force (RCF) intensity on LTA results. METHODS: Ten healthy controls had venous blood drawn and centrifuged at 150, 200, 300, and 500 g for 10 min. Cell counts in whole blood and platelet-rich plasma (PRP) were measured using a hematology analyzer. LTA was performed using 1.0 µm adenosine diphosphate (ADP) and 0.4 µm epinephrine as an agonist. Aggregation (%) was compared at 60, 120, 180, and 300 s and at maximum aggregation. RESULTS: Centrifugation speed was associated with decreasing platelet count (P < 0.001) and decreasing mean platelet volume (P < 0.001) in PRP. Maximum aggregation decreased with increasing speeds for ADP 1.0 µm (150 g- 89%, 200 g- 93%, 300 g- 71%, 500 g- 17%; P < 0.001). Similar findings were noted at 120 s (150 g- 69%, 200 g- 50%, 300 g- 35%, 500 g- 12%; P < 0.001), 180 s (150 g- 82%, 200 g- 74%, 300 g- 44%, 500 g- 13%; P < 0.001), and 300 s (150 g- 85%, 200 g- 88%, 300 g- 55%, 500 g- 14%; P < 0.001). Consistently, platelet aggregation in response to epinephrine 0.4 µm decreased significantly with increasing centrifuge RCF at 60, 120, 180, 300 s and at maximum aggregation (P < 0.05 for each comparison). CONCLUSION: Our data demonstrate the importance of centrifugation speed in the interpretation of LTA results, supporting the need for standardization of centrifugation RCF in LTA protocols.

HIV-1 Tat-induced platelet activation and release of CD154 contribute to HIV-1-associated autoimmune thrombocytopenia.

BACKGROUND: Enhanced platelet activation in human immunodeficiency virus (HIV)-1-infected patients has been reported and shown to strongly correlate with plasma viral load. Activated platelets are known to express and to release a variety of proteins that can modulate the immune system. Specifically, platelet-derived CD154 has been shown to be directly involved in the development of autoimmune thrombocytopenia (ITP). The mechanism by which HIV-1 infection leads to platelet activation and the effect of this activation on the development of HIV-1 ITP, however, is not fully understood. OBJECTIVE: We have investigated the effect of HIV-1 Trans activating factor (Tat) on platelet activation. RESULTS: We report that HIV-1 Tat directly interacts with platelets and induces platelet activation resulting in platelet micro-particle release. This activation by Tat requires the chemokine receptor CCR3 and ß3-integrin expression on platelets, as well as calcium flux. Tat-induced activation of platelets releases platelet CD154, an immune modulator. Enhanced B-cell activity is found in mouse spleen B cells co-cultured with platelets treated with Tat in vitro. An early antibody response against adenovirus is found in Tat-injected mouse immunized with adenovirus, suggesting an enhanced immune response in vivo. CONCLUSIONS: We have described a role of Tat-induced platelet activation in the modulation of the immune system, with implications for the development of HIV-1-associated thrombocytopenia.

GPIIIa-(49-66) is a major pathophysiologically relevant antigenic determinant for anti-platelet GPIIIa of HIV-1-related immunologic thrombocytopenia.

High-affinity (Kd = 1 x 10(-9) M) anti-platelet GPIIIa has been isolated from serum immune complexes of immunologic thrombocytopenic HIV-1-infected patients (HIV-1-ITP). Affinity-purified anti-platelet antibody reacted with a recombinant GPIIIa-(1-200) and -(1-66) fusion peptide and with an 18-mer GPIIIa-(49-66) peptide but not with seven other GPIIIa peptides spanning the length of GPIIIa. Most of the anti-platelet antibody ( approximately 85%) could be adsorbed to and eluted from a GPIIIa-(49-66) affinity column. Binding of antibody to platelets could be inhibited by GPIIIa-(49-66) or an equimolar peptide-albumin conjugate (IC50 = 2 microM). Sera from 7 control subjects and 10 classic autoimmune thrombocytopenic patients gave background reactivity with GPIIIa-(49-66). HIV-1-ITP sera from 16 patients reacted with a mean OD 6-fold greater than background (range, 4- to 9-fold). Serum anti-GPIIIa-(49-66) concentration correlated inversely with platelet count, R2 = 0.51, n = 31, P < 0. 0001. Because mouse platelet GPIIIa-(49-66) has 83% homology with human GPIIIa and mouse monocytes contain Fc receptors for the human IgG1-kappa/lambda antibody, we determined the in vivo effect of human anti-GPIIIa on mouse platelets. Affinity-purified antibody, 25-50 microg given i.p., resulted in a precipitous drop in platelet count to 30% of baseline, with nadir at 4 hr and return to normal in 36 hr. No effect was noted with control IgG. Acute thrombocytopenia could be prevented or reversed by the injection of the GPIIIa-(49-66) albumin conjugate at zero time or 2 hr after antibody, respectively, but not with a scrambled peptide-albumin conjugate. Thus HIV-1-ITP patients have high-affinity anti-platelet GPIIIa against a major antigenic determinant, GPIIIa-(49-66), which correlates inversely with platelet count and induces thrombocytopenia in mice.

Inhibition of binding of anti-PLA1 antibodies to platelets with monoclonal antibody LK-4. Evidence for multiple PLA1 receptor sites on platelet GPIIIa.

The PLA1 epitope on platelet GPIIIa has a sulfhydryl-dependent conformation and is dependent on a leucine 33/proline33 polymorphism. Monoclonal antibody LK-4 differentiates PLA1/PLA1 from PLA2/PLA2 platelet lysates on solid phase enzyme-linked immunosorbent assay (ELISA), as well as immunoblot. To determine whether LK-4 reacts at or near the binding site(s) for human anti-PLA1, nine such antibodies (Abs) (six neonatal; three posttransfusion) were examined in the presence and absence of LK-4 for binding to platelets, as well as rGPIIIa 1-66, a recombinant glutathione S-transferase fusion peptide. All nine human Abs bound to rGPIIIa 1-66, as well as platelets, in a saturation-dependent manner, employing both solid phase ELISA, as well as flow cytometry. Binding of all nine Abs to rGPIIIa 1-66 or platelets was inhibited by LK-4. IC50's for inhibition of binding of anti-PLA1 to rGPIIIa 1-66 varied from 8 to 160 micrograms/mL (5 x 10(-8)- 1 x 10(-6) mol/L). However, IC50's for LK-4 inhibition of binding to platelets was strikingly different. Six of the nine Abs had IC50's of 1 to 10 micrograms/mL (8-fold to 16-fold greater inhibition than with rGPIIIa 1-66), whereas three neonatal Abs had IC50's of 380 to 1,013 micrograms/mL (6-fold to 48-fold less inhibition than with rGPIIIa 1-66). Similar results were noted with intact GPIIIa, rGPIIIa 1-66 blocked the binding of anti-PLA1 Abs to platelets and served to segregate the nine patients into two groups: a sensitive group of anti-PLA1 Abs from six patients in which binding to platelets was progressively inhibited by increasing concentrations of rGPIIIa 1-66 with inhibition at 1 micrograms/mL of 18% and inhibition at 256 micrograms/mL of 78%; a second resistant group of three anti-PLA1 Abs from three patients in which inhibition was first noted at 16 micrograms/mL of 4% with 35% inhibition at 256 micrograms/mL. Thus, LK-4 binds to GPIIIa at the 1-66 N-terminal region, inhibits binding of anti-PLA1 Ab to platelets, and segregates, anti-PLA1 Abs into two groups. These data are compatible with two or more receptor sites for anti-PLA1 Ab: one that is present on rGPIIIa 1-66 and sensitive to LK-4 inhibition, another that is present on rGPIIIa 1-66, as well as other site(s) on platelet GPIIIa and insensitive to inhibition.

Transient hemorrhagic diathesis associated with an inhibitor of prothrombin with lupus anticoagulant in a 1 1/2-year-old girl: report of a case and review of the literature.

Acquired inhibitors of coagulation causing bleeding manifestations are rare in children, particularly without an associated underlying disorder such as autoimmune disease. We describe an otherwise healthy 1 1/2-year-old girl who had extensive spontaneous bruising and prolonged bleeding from venipuncture sites. Prothrombin time (PT) and activated partial thromboplastin time (APTT) were prolonged, with evidence of an immediate-acting inhibitor. Thrombin clotting time, fibrinogen, and platelets were normal. Biologic assay of factors II, V, VII, and X were all low, with increasing values at higher dilutions. However, by immunoassay and/or chromogenic assays, only factor II was reduced. An antibody which failed to neutralize prothrombin activity in vitro was detected against radiolabeled prothrombin. Coagulation studies normalized in parallel with clinical recovery and disappearance of the antibody. This case demonstrates acute hypoprothrombinemia-lupus anticoagulant syndrome as a rare presentation of bleeding diathesis in a healthy young child.

Heterogenous inhibition of platelet aggregation by monoclonal antibodies binding to multiple sites on GPIIIa.

Six monoclonal IgG1-k antibodies (LK2, LK3r, LK4-55, LK5, LK6-55, LK7r) were raised against platelet membrane GPIIIa in order to study the structure-function relationship of this molecule. Antibodies were selected on their ability to react with GPIIIa by ELISA on adherent platelets, by immunoblot on platelet lysates and by fluorescence flow cytometry on intact platelets. Fluorescence reactivity varied from 3- to 202-fold greater than isotype control fluorescence. Two MoAbs reacted on immunoblot under reduced conditions (LK7r and LK3r). Two reacted with a 55 kD chymotrypsin/subtilisin digest of GPIIIa which is likely to exclude amino acids 121-348 (LK4-55 and LK6-55). Four of the MoAbs (LK5, LK3r, LK2 and LK4-55) inhibited tyrosine phosphorylation of one to four distinct bands on immunoblot. LK4-55 reacted with an N-terminal 66 amino acid fusion protein of GPIIIa near the PLA epitope (Leu 33). LK7r reacted with a 212-222 peptide reported to be an RGD fibrinogen binding site. LK2 reacted near a disintegrin-RGD binding site. Except for LK5, all inhibited ADP, collagen and thrombin-induced platelet aggregation in a heterogeneous fashion. Percentage inhibition of 125I-fibrinogen binding to platelets varied from 18% to 98%. No correlation was noted between inhibition of fibrinogen binding, location of MoAb binding on GPIIIa, reactivity of MoAb binding with GPIIIa, inhibition of thrombin-induced tyrosine phosphorylation or inhibition of platelet aggregation induced by ADP, collagen or thrombin. Thus MoAbs, binding to platelet GPIIIa at different sites, inhibit platelet aggregation in a heterogeneous manner.

Production of a human anti-CD4 monoclonal antibody with antiidiotype to anti-HIV type 1 glycoprotein 120.

Human anti-CD4 IgG antibodies from 3 HIV-1-infected patients were affinity purified and shown to inhibit HIV-1 binding and infection of HBP-T cells. Lymphocytes from patient A, whose anti-CD4 inhibited HIV-1 binding by 68% and infection by 72%, were cultured and transformed with EBV. A human monoclonal antiidiotype antibody against anti-HIV-1 gp120 (2B) was produced, which inhibited infection of HBP-T cells by 68% at 1 microgram/ml. Mice were immunized with 2B to determine whether this anti-CD4 could be an internal image antiidiotype against anti-HIV-1 gp 120 (Ab1). Two mice produced antisera reactive with rgp120 on ELISA, whereas immunization with normal IgG produced minimal reactivity compared to unreactive normal mouse sera. However, immunoblot competition studies in which affinity-purified anti-HIV-1 gp120 (Ab1) bound to the gp120 band on nitrocellulose strips in the presence of 2B demonstrated enhancement of signal (i.e., binding of Ab2 to Ab1), rather than inhibition of Ab1 binding. Thus 2B is not an internal image of the paratope of anti-HIV-1 gp120 but yet it is capable of inducing an antibody against rgp120. This indicates that the anti-CD4 (Ab2) does bind to the binding site of Ab1, but not as a complete internal image. These data indicate the production of a human monoclonal antiidiotype antibody that inhibits binding of HIV-1 to CD4 and induces the production of antibody against HIV-1 gp120, without being an internal image antiidiotype (Ab2 beta).

Sequestration of anti-platelet GPIIIa antibody in rheumatoid factor immune complexes of human immunodeficiency virus 1 thrombocytopenic patients.

Human immunodeficiency virus 1-related idiopathic thrombocytopenic purpura (HIV-1-ITP) patients have a 4-fold increased percentage of CD5+ B cells and a 4.8-fold increased percentage of serum immune complexes precipitated by polyethylene glycol (PEG-ICs) compared to control subjects, as reported previously. Since CD5+ B cells produce predominantly IgM rheumatoid factor (RF) vs. Fc of IgG and PEG-ICs contain high levels of IgM, we looked for the presence of RF in the immune complexes of HIV-1-ITP patients. PEG-ICs were adsorbed to protein A and dissociated with acid, and IgM and IgG were purified by gel filtration and affinity chromatography. Solid-phase ELISA was used to measure antibody specificity vs. platelets, Fc, and HIV-1 gp120, p24, and CD4. Dissociated IgG antibody reacted with platelets, HIV-1 gp120, p24, and CD4, but not with Fc. Serum IgG did not react with platelets or Fc but did react with HIV-1 gp120, p24, and CD4. Both PEG-IC IgM and serum IgM reacted with Fc as well as the other four antigens. Control IgM and IgG were unreactive. Isolated IgM from PEG-ICs relocated approximately 50% of the IgG preincubated with IgM to the Vo region of a G200 gel-filtration column. Anti-platelet IgG but not IgM could be affinity-purified from fixed platelets. Both F(ab')2 fragments of anti-platelet IgG and the total PEG-IC bound to platelets in a saturation-dependent manner. F(ab')2 of anti-platelet IgG inhibited 50% binding of PEG-IC to platelets at an F(ab')2/complex ratio of 3:1 (wt/wt). Scatchard analysis revealed two classes of binding sites: high-affinity Kd values of 0.8-1.8 nM and lower-affinity Kd values of 6.6-12.3 nM with respective numbers of binding sites of 44,000-57,000 and 122,000-256,000 (n = 4). Anti-platelet IgG of 6/6 patients precipitated GPIIIa from platelet lysates of surface 125I-labeled platelets. Platelet count correlated inversely with anti-platelet IgG (r = -0.73; P < 0.01; n = 27). Thus, PEG-ICs of HIV-1-ITP patients contain IgM RF, which sequesters serum anti-platelet IgG containing anti-GPIIIa. Anti-platelet IgG contributes to binding of immune complexes to platelets and correlates with thrombocytopenia.

A monoclonal antibody (LK-4) which differentiates PLA1 from PLA2 platelet extracts but not intact platelets.

A unique murine monoclonal antibody (LK-4) is described which differentiates PLA1/PLA1 platelet extracts from PLA2/PLA2 and PLA1/PLA2 platelet extracts on solid phase ELISA and immunoblot at the 100kD GPIIIa location, but not on intact platelets. LK-4 reacts equally with intact PLA1/PLA2 and PLA2/PLA2 platelets. Adsorbtion of LK-4 with PLA1/PLA1 platelets results in loss of reactivity for intact platelets as well as platelet extracts on ELISA or immunoblot. LK-4 inhibits platelet aggregation induced by ADP, epinephrine, collagen and thrombin, suggesting reactivity at or near the fibrinogen binding site on GPIIIa. It is suggested the LK-4 reacts with a conformation-induced common epitope for PLA1 and PLA2 on GPIIIa, with loss of this conformation for PLA2 GPIIIa following solubilization with Triton X-100.

Internal-image anti-idiotype HIV-1gp120 antibody in human immunodeficiency virus 1 (HIV-1)-seropositive individuals with thrombocytopenia.

Anti-CD4 antibody was found in 30% of human immunodeficiency virus (HIV-1)-seropositive thrombocytopenic patients compared with 5% of nonthrombocytopenic seropositive patients (chi 2 = 21.7, P less than 0.001) and was shown by the following observations to contain internal-image anti-idiotype antibody (Ab2) directed against the antibody (Ab1) to gp120, the HIV-1 envelope glycoprotein that binds to CD4: (i) affinity-purified anti-CD4 (Ab2) bound to affinity-purified anti-HIV-1gp120 (Ab1) on solid-phase radioimmunoassay, and binding could be blocked by recombinant CD4 (rCD4) as well as recombinant gp120 (rgp120); (ii) F(ab')2 fragments of Ab1 inhibited the binding of Ab2 to rCD4; (iii) Ab2 inhibited the binding of Ab1 to HIV-1 beads; (iv) Ab2 inhibited the binding of Ab1 to gp120 on immunoblot; (v) Ab2 bound to the CD4 receptor on a CD4-bearing T-cell line, H9; (vi) Ab3 (anti-rgp120) could be produced in vivo by immunizing mice with Ab2, and binding of Ab3 to rgp120 could be blocked with rCD4; and (vii) three different Ab2 preparations bound to two different homologous Ab1 preparations. Ab1 or Ab2 alone did not bind to platelets, whereas the idiotype-anti-idiotype complex did bind to platelets in a concentration-dependent manner. Binding of the internal-image complex was 10-fold greater than that of a non-internal-image Ab1-Ab2 complex composed of anti-HIV-1gp120 and anti-anti-HIV-1gp120. Thus, patients with HIV-1 thrombocytopenia contain internal-image idiotype-anti-idiotype complexes that could be affecting CD4 cell number or function, inhibiting HIV-1 binding to CD4 cells or contributing to HIV-1 thrombocytopenia.

Immunologic thrombocytopenic purpura after heterosexual transmission of human immunodeficiency virus (HIV).

We report four cases of immunologic thrombocytopenic purpura related to human immunodeficiency virus (HIV) transmitted through heterosexual contact in persons who were not homosexual, addicted to intravenous narcotic drugs, or hemophilic. Each transmission occurred in a different setting. A 23-year-old white woman had immunologic thrombocytopenic purpura in July 1985, with a platelet count of 11 X 10(9)/L. In January 1987, she had prominent submandibular and posterior cervical adenopathy. A careful social-sexual history revealed several sexual contacts with a male narcotic addict before July 1985. A 27-year-old heterosexual white man had a platelet count of 8 X 10(9)/L in December 1986. A social-sexual history revealed that his fiancée had been an intravenous narcotic addict 6 years ago. A 64-year-old white woman had a platelet count of 75 X 10(9)/L in May 1986, approximately 2 years after she had resumed having sexual intercourse with her husband who had had a triple coronary bypass in October 1983. The husband had received HIV-seropositive blood. A 42-year-old white man had a platelet count of 45 X 10(9)/L, which was associated with a cutaneous eruption refractory to antibiotics and antifungal agents. He had had sexual contacts with several women, who, to the best of his knowledge, were neither prostitutes nor intravenous narcotic addicts. He denied homosexuality or drug abuse. All four patients were HIV-seropositive and had circulating immune complexes and platelet-associated IgG, C3C4, and IgM values that were considerably higher than those usually measured in patients with classic autoimmune thrombocytopenia, averaging 2.4-, 2.2-, 6.5- and 5.2-fold higher, respectively. Thus, HIV-related immunologic thrombocytopenic purpura can be heterosexually spread and should become part of the differential diagnosis of unexplained thrombocytopenia. Obtaining a careful social-sexual history is mandatory in such patients.

Immunologic thrombocytopenic purpura in human immunodeficiency virus--seropositive patients with hemophilia. Comparison with patients with classic autoimmune thrombocytopenic purpura, homosexuals with thrombocytopenia, and narcotic addicts with thrombocytopenia.

The immunologic platelet profile of 29 patients with hemophilia who had received multiple transfusions (14 had thrombocytopenia) was compared with profiles of 15 patients with classic autoimmune thrombocytopenia. Thrombocytopenic hemophiliacs who were seropositive for the human immunodeficiency virus (13 out of 14) had platelet-bound immunoglobulin G and C3C4 levels as well as circulating immune complexes that were 15.1-, 4.0-, and 2.4-fold greater, respectively, than normal control subjects' platelets and 3.4-, 2.6-, and 2.4-fold greater, respectively, than autoimmune thrombocytopenic patients' platelets. Hemophiliacs with normal platelet counts (nine out of 13 seropositive) had elevated values that were intermediate in level between those of thrombocytopenic hemophiliacs and those of classic autoimmune thrombocytopenic patients. The four seronegative hemophiliacs had normal values (except for one platelet-bound IgG measurement). An inverse correlation was noted between platelet count and platelet-bound IgG, r = -0.838, P less than 0.001. Serum antiplatelet reactivity (1:256 mean titer compared with control sera) resided predominantly in the 7S IgG fraction, and bound to autologous as well as homologous platelets at concentrations as low as 0.06 to 0.13 mg/ml. F(ab')2 fragments of the 7S IgG fraction inhibited binding of hemophilic IgG to normal platelets and bound to normal platelets at concentrations as low as 0.06 mg/ml. Antiplatelet IgG could be eluted from six of six hemophilic and eight of eight classic autoimmune thrombocytopenic patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Thrombocytopenic purpura in narcotics addicts.

Since November 1982 we have seen an association of thrombocytopenic purpura with chronic narcotic addiction in 70 patients with a mean platelet count of 53000 +/- 4000 (SE); 33 had stopped taking intravenous drugs for an average of 21.2 +/- 4.7 months; 13 of 15 had elevated antibody titers for a virus related to the acquired immunodeficiency syndrome. Platelet-bound IgG, IgM, and complement levels were 16.7-, 5.6-, and 3.1-fold greater than control values, respectively, and 2.6-, 1.9-, and 2.4-fold greater than values in 25 patients with classic autoimmune thrombocytopenic purpura studied at the same time. Thirty-three of thirty-six addicts had elevated circulating immune complexes, whereas 8 patients with autoimmune thrombocytopenia had no elevation. Eleven of eighteen addicts had positive serum platelet-reactive IgG titers, compared to 5 of 19 patients with classic autoimmune thrombocytopenia. The platelet-reactive IgG in sera of addicts was composed of 7S IgG antibody as well as high molecular weight (immune complex) IgG. Thus, chronic addicts appear to have a new immunologic platelet disorder associated with the presence of 7S IgG antiplatelet antibody, like patients with classic autoimmune thrombocytopenic purpura, and immune complex associated nonspecific platelet IgG, like male homosexual patients with thrombocytopenia.

On the mechanism of thrombocytopenic purpura in sexually active homosexual men.

Thrombocytopenic purpura has recently been noted in sexually active homosexual men. To elucidate the pathogenesis of thrombocytopenic purpura in this population, we compared the disorder in 33 homosexual men with that in 23 patients (15 women and 8 men) thought to have classic autoimmune thrombocytopenic purpura. The homosexual group had 3.8-fold higher levels of platelet-bound IgG and 4.2-fold higher levels of platelet-bound complement than the patients with autoimmune thrombocytopenic purpura. Eluates from the platelets of only 1 of 10 homosexual patients reacted in vitro with normal platelets, as compared with those from the platelets of 12 of 15 patients with autoimmune thrombocytopenic purpura. Twenty-one of 24 homosexual patients (88 per cent) had elevated serum levels of immune complexes that were capable of binding to platelets, whereas none of 5 patients with autoimmune thrombocytopenic purpura had circulating immune complexes. The IgG fraction of positive serum samples from three homosexual patients did not bind to normal platelets, whereas that from the positive serum of two patients with autoimmune thrombocytopenic purpura and one woman in whom isoimmune antiplatelet antibody developed during pregnancy (studied as a positive control) did bind to normal platelets. We conclude that, whereas classic autoimmune thrombocytopenic purpura involves antiplatelet IgG directed against platelet antigenic determinants, the thrombocytopenic purpura that occurs in sexually active homosexual men is usually caused not by antiplatelet IgG but probably by the nonspecific deposition of complement and immune complexes on platelets.

Leukocyte-platelet interactions in a murine model of Chediak-Higashi syndrome.

Chediak-Higashi (CH) syndrome, a genetic disease affecting man and other animals, is partially characterized by defective platelets that lack serotonin and dense bodies and by impaired leukocyte function where chemotaxis, degranulation, and bacterial killing are decreased. The effects of normal platelets containing serotonin and of reagent serotonin on the subnormal microbicidal activity of CH leukocytes were evaluated. The peripheral blood leukocytes of the beige mouse, an animal model with CH syndrome, were used with Staphylococcus aureus as the bacterial challenge. Addition of as few as two normal platelets/leukocyte resulted in normal levels of microbicidal activity of CH leukocytes. A similar normalization of leukocyte function was seen when 1-100-micrometer serotonin was added to the incubation mixture. Based on this work and work of others, a plausible explanation for these observations is that normal platelets interact with CH leukocytes, releasing serotonin, which results in reversal of the CH leukocyte defect in bacterial killing.