Thrombotic thrombocytopenic purpura (TTP) and hemolytic uremic syndrome (HUS): the new thinking.

TTP and HUS are two disorders with many similarities. Though their first descriptions appeared at different time in history, there has been a trend among physicians to consider them as the same clinical entity. However, in recent years new research findings on the pathophysiology of TTP and HUS have revealed some differences between the two disorders. In this paper, we will review the current approaches to the clinical and laboratory diagnosis of TTP and HUS, as well as therapeutic strategies. We will also summarize the recent advances in three areas in the study of the pathophysiology of TTP and HUS, namely the newly discovered von Willebrand factor multimer-cleaving protease, endothelial cell apoptosis induced by serum from patients with TTP and atypical HUS and the activation of complement system. Since distinguishing and differentiating between TTP and HUS may help to develop more effective therapies targeted at key steps of the disease development, we will discuss possible ways of reclassifying the TTP-HUS disorders. In the end, we also present our views on possible future development.

Expression of translation initiation factors elF-4E and elF-2alpha and a potential physiologic role of continuous protein synthesis in human platelets.

It is generally believed that platelets do not have a functionally significant protein synthetic machinery. However, our analysis demonstrated that normal bone marrow megakaryocytes express high levels of translation initiation factors eIF-4E and eIF-2alpha and the expression of these protein synthesis initiation factors is continued in platelets (as determined by immunohistochemistry and Western blot analysis). Both eIF-4E and eIF-2alpha are key regulators of protein synthesis. The eIF-4E is a rate-limiting part of a multisubunit complex, eIF-4F, that binds to the 5' cap structure present in virtually all eukaryotic mRNAs, and carries out transfer of mRNAs to ribosomes for translation. Translation initiation factor eIF-2alpha is also a rate-limiting protein which associates with two other proteins to form an eIF-2 initiation factor complex responsible for the transfer of initiator methionyl-tRNA to the 40S ribosomal subunit. We confirm that expression of eIF-4E and eIF-2alpha is biologically relevant in that platelets continue protein synthesis, albeit at a 16 times lower rate than WBC (as determined by 35S-labeled amino acid incorporation, SDS-PAGE and scintillation counting). Finally, we determined that protein synthesis inhibitors (puromycin and emetine) attenuate the platelet aggregation response to a combination of ADP and epinephrine, but potentiate the response to collagen. Our data are consistent with the existence of different signal transducing pathways mediating the response to ADP/epinephrine and collagen. We suggest that the ADP/epinephrine response is positively affected by continuously synthesized proteins, while the response to collagen is modulated by continuously produced inhibitory proteins. Taken together, our results suggest that continuous protein synthesis is important for platelet function and its role in platelet physiology and pathophysiology deserves further study.

Screening tests of disseminated intravascular coagulation: guidelines for rapid and specific laboratory diagnosis.

OBJECTIVE: To study the clinician's ordering pattern in the diagnosis of disseminated intravascular coagulation (DIC) and to analyze the utility of selected tests by assessing their sensitivity, specificity, and overall efficiency. DESIGN: Retrospective, nonrandomized, clinical study. SETTING: University hospital intensive care units. PATIENTS: A total of 82 inpatients treated in our intensive care units were identified from the hospital computer system as having been tested for DIC in a 3-month period. INTERVENTION: Screening tests for DIC were ordered for the suspected patients. MEASUREMENTS AND MAIN RESULTS: Prothrombin time (PT), partial thromboplastin time (PTT), fibrinogen/fibrin degradation products (FDP), and fibrinogen were used most frequently as DIC diagnostic tests. The FDP and D-dimer combination (n = 39) had the highest diagnostic efficiency of 95%, with sensitivity being 91% and specificity 94%. This is followed by FDP (n = 71), efficiency 87%, sensitivity 100%, and specificity 67%; PT/PTT and FDP combination (n = 71), efficiency 86%, sensitivity 91%, and specificity 71%; and D-dimer (n = 44), efficiency 80%, sensitivity 91%, and specificity 68%. The rest of the commonly used tests, such as PT, PTT, thrombin time, platelet count, fibrinogen, and the presence of schistocytes (n = 80), had individually either low specificity or low sensitivity and, therefore, low efficiency scores (57%, 57%, 70%, 67%, 65%, and 51%, respectively). CONCLUSIONS: The D-dimer and FDP tests offered the best test panel in the diagnosis of DIC. We propose the use of D-dimer, FDP, and antithrombin as the DIC diagnostic test panel, with D-dimer and FDP providing a rapid and specific diagnosis, antithrombin providing insight to the severity and prognosis, and FDP (rapid and less expensive than D-dimer) to follow-up the progress of the condition once the diagnosis is established.

Clinical implications of elevated PAI-1 revisited: multiple arterial thrombosis in a patient with essential thrombocythemia and elevated plasminogen activator inhibitor-1 (PAI-1) levels: a case report and review of the literature.

Plasminogen activator inhibitor (PAI-1), a member of the serine protein family, is the most active in vivo inhibitor of fibrinolysis induced by plasminogen, tissue plasminogen activator (tPA), and urokinase type plasminogen activator (uPA). While the association between elevated PAI-1 and thrombogenesis has been well studied for several disease processes, including coronary disease, postoperative deep vein thrombosis (DVT), myocardial infarction, malignancy, and diabetes, few studies have concentrated on the correlation between elevated PAI-1 levels and thrombogenesis in patients with myeloproliferative disorders. Essential thrombocythemia (ET), a chronic myeloproliferative disorder, characterized by the overproduction of poorly functioning platelets, is associated with both thrombotic and hemorrhagic life-threatening complications. Although the events resulting in thrombogenesis in such patients may be multifactorial in nature, an association between elevated PAI-1 levels and thrombus formation has been proposed. Herein we present a patient diagnosed with ET complicated by multiple episodes of arterial thrombosis. Elevations in PAI-1 levels were documented repeatedly. The role of elevated PAI-1 when associated with other disease processes is also discussed.

A diagnostic dilemma: chronic myelomonocytic leukemia versus atypical chronic myeloid leukemia. A case report and review of the literature.

There exists a great deal of overlap between many myelodysplastic syndromes and myeloproliferative disorders. This is most evident in the spectrum of disorders classified under the term chronic myeloid leukemia. These include chronic granulocytic leukemia, atypical chronic myeloid leukemia and chronic myelomonocytic leukemia. Current classification often does not clearly separate these entities since they share many features, both clinically and hematologically. We report here a case that satisfies criteria for both chronic myelomonocytic leukemia and atypical chronic myeloid leukemia, appearing to fluctuate between the two. This lends further evidence for the heterogeneity of these disorders and the need for better definition. An improved classification scheme would allow for more accurate reporting and research into etiology and treatment. The complex cytogenetic abnormalities of the case are unique and to our knowledge have not been reported previously. Also, this case report underscores the importance of cytochemical stains when such disorders are under consideration.

Leukemia with megakaryocytic differentiation following essential thrombocythemia and myelofibrosis. Case report and review of the literature.

Leukemias of megakaryocytic lineage are rare and heterogeneous clinical entities. The nomenclature published in the literature is confusing and perhaps inappropriate to designate these primary myeloproliferative disorders. We describe a patient with essential thrombocythemia who evolved through myelofibrosis and myeloid metaplasia to a final picture of leukemia with megakaryocytic differentiation in the peripheral blood. This case illustrates different aspects of a chronic myeloproliferative disorder where myelofibrosis and myeloid metaplasia are frequent but secondary events. We have reviewed the literature focusing on the role of clonal megakaryocytic proliferation in myelofibrosis and on the clinical characterization of leukemia with megakaryocytic phenotype. We also present our interpretation of the literature which indicates that a formal review of the nomenclature is urgently needed.

Continuous improvement, quality control, and cost containment in clinical laboratory testing. Effects of establishing and implementing guidelines for preoperative tests.

OBJECTIVE: To develop guidelines for laboratory tests ordered before admission for elective surgery. DESIGN: A seven-step continuous quality improvement process. SETTING: The Departments of Laboratory Medicine, Surgery, and Anesthesia of the University of Massachusetts Medical Center, a 384-bed, teaching, tertiary-care facility. PARTICIPANTS: Core group of the Department of Laboratory Medicine and the Laboratory Medical Advisory Committee. INTERVENTION: Guidelines were developed for laboratory tests ordered before elective surgery. They were divided into four major groups as well as by age and gender. After an intense educational effort, consent was obtained from the majority of surgeons, who agreed to delegate the ordering of tests to the nurses and anesthesiologists who examine patients before surgery. MAIN OUTCOME MEASURE: Charts chosen at random by the medical records department for the period prior to implementation of guidelines were reviewed and compared with records 1 and 2 years later. RESULTS: Reductions of 50% and 60% in the first and second years, respectively, in the overall number of tests ordered per patient were demonstrated. An improvement in the appropriateness of tests was also documented: 81% in the first year and 86% in the second year, compared with 65% appropriateness prior to implementation of guidelines. A 1-year savings of $66,981 and an overall 2-year savings of $75,995 were documented. CONCLUSIONS: We have described an approach that involves a sustained educational effort and collaboration of nurses and physicians and have presented specific guidelines for preoperative testing. A major decrease in the number of tests ordered, an increase in their appropriateness, and marked fiscal savings were documented.

A laboratory rotation for medical house officers. Bridging the gap.

In an attempt to improve physicians' laboratory practice behavior, the Department of Hospital Laboratories at the University of Massachusetts Medical Center developed a rotation for first year housestaff. Medical interns were chosen for this pilot program because they are the most frequent users of our laboratory facilities. Rotations provide an overview of the laboratory organization, quality control and assurance, appropriate use of laboratory testing, cost containment, and an introduction to different laboratory disciplines. As assessed by discussions during an interview following completion of the program, the participants have shown an increased understanding of how a modern hospital laboratory functions and of the complexity of services provided. The respect for the laboratory staff and confidence in test results issued have increased, and house officers are more likely to use laboratory services in a more cost-efficient manner.

Continuous improvement, quality control, and cost containment in clinical laboratory testing. Enhancement of physicians' laboratory-ordering practices.

In 1991, the University of Massachusetts Medical Center, in Worcester, developed a model for change by using a program of continuous quality improvement to enhance physicians' laboratory-ordering practices, particularly the test for bleeding times. We describe a model that was developed through a seven-step continuous quality improvement process, and we discuss our success in increasing the appropriateness of ordering the tests for bleeding times while significantly reducing the costs for patients and hospitals. The following factors contributed to the program's success: an advisory structure; presentations to the medical staff; focused feedback sessions; and most important, well-documented guidelines with institutional support for new behavior.

Hypothesis for the control of clotting factor VIII inhibitory antibodies by decreasing potency of helper T-cell-recognized epitopes in factor VIII.

The study of the immunobiology of FVIII inhibitors may lead to new therapies for this potentially severe complication of haemophilia A and to new principles for the use of therapeutic proteins. In order to characterize the idiotype-anti-idiotype networks regulating FVIII inhibitors, we developed rabbit anti-idiotypic sera to 7 murine inhibitors and found at least 12 independent FVIII loci to which inhibitors could be raised. Rabbit antisera to the FVIII peptide, Ser1687-Thr1695, characterized one functional site to which about 46% of patients' inhibitor sera reacted. The multiplicity of inhibitor-recognized epitopes in FVIII makes it impractical, at the present time, to develop clinically useful specific anti-idiotypic therapies for FVIII inhibitors. Alternatively, one might induce genomic mutations in recombinant FVIII molecules to decrease immunogenicity of epitopes recognized by T helper cells. Methods to design such altered therapeutic proteins are presented, based on changing the longitudinal hydrophobic strip-of-helix which is in or near many T-cell-presented epitopes.

Characterization of a factor VIII immunogenic site using factor VIII synthetic peptide 1687-1695 and rabbit anti-peptide antibodies.

A 9 amino acid peptide, Ser-Pro-Arg-Ser-Phe-Gln-Lys-Lys-Thr, corresponding to the clotting factor VIII (FVIII) sequence Ser1687-Thr1695, was synthesized in order to analyze a site on FVIII to which antibody inhibitors of FVIII may be directed. This sequence contained a thrombin cleavage site. It was predicted to be immunogenic because a Hopp-Woods hydrophilicity analysis of the amino acid sequence of FVIII showed it to be very hydrophilic, and it contained a proline. The HPLC-purified peptide was cleaved by thrombin at Arg1689-Ser1690, as determined by amino acid sequencing of the cleavage product. Thrombin which had been treated with a specific chloromethyl ketone inhibitor, did not cleave the peptide. Two rabbits immunized with the peptide/keyhole limpet hemocyanin conjugate generated FVIII inhibitory sera with titers of 5.4 and 4.8 Bethesda units. These rabbit anti-peptide antibodies reacted with a peptide/-BSA conjugate on immunodot blot analyses and with native, affinity-purified FVIII in Western blots. In competitive immunoradiometric assays, cryosupernatants of 38/82 patients with FVIII inhibitors reacted with the synthetic peptide. We conclude that FVIII peptide Ser1687-Thr1695 is cleaved by thrombin at the same peptide bond which is cleaved in FVIII, and the peptide contains a site to which patients' inhibitory antibodies can be directed.

Factor VIII epitopes recognized with inhibitory monoclonal antibodies.

To test whether anti-idiotypic immunoregulation of factor VIII(FVIII)-inhibiting antibodies could be feasible in hemophiliacs, we assayed the minimal number and range of immunogenic, functional FVIII epitopes to which a series of murine anti-FVIII monoclonal antibodies (MAb) were directed. Rabbit anti-idiotypic sera to 7 MAb were prepared and used in competitive immunoradiometric assays to test Id similarities among 36 murine MAb. At least 12 immunogenic and functional epitopes were found on the FVIII molecule. The large range of target loci for FVIII inhibitors will make their immunoregulation by anti-idiotypic methods difficult.

Development of anti-idiotypic antibodies in a patient with a factor VIII autoantibody.

Antibodies against coagulation factor VIII were detected in a 33-year-old who developed severe bleeding 2 months postpartum. After treatment with plasmapheresis and immunosuppressive agents, the inhibitor was no longer detectable, but anti-idiotypic antibodies were detected, as demonstrated by binding in immunoradiometric assays, and by in vitro neutralization of the initial factor VIII autoantibodies. The anti-idiotypic titer subsequently declined but was still detectable 1 year later. Three commercial immunoglobulin preparations and pooled multiparous IgG also bound to (but did not neutralize) the patient's factor VIII antibody. These studies suggest that anti-idiotypic antibodies, arising in the face of immunosuppressive therapy, might suppress autoimmunity to factor VIII and confirm the presence of anti-idiotypic antibodies in pooled normal immunoglobulins.

Identification of six functional clotting factor VIII:C epitopes by analysis of cross-reactive public idiotypes in murine monoclonal VIII:C inhibitors.

Six factor VIII:C epitopes which can elicit clotting factor inhibitory antibodies were demonstrated by analysis of public idiotypes of murine monoclonal anti-VIII:C inhibitors. Anti-idiotypic immunoradiometric assays were developed with rabbit antibodies to murine VIII:C inhibitors: Synbiotics, Hybritech, C7F7 and RFF-VIIIC/8. Crossreactions among 10 murine monoclonal antibodies (MoAbs) to VIII:C were tested, showing 6 functional and immunospecific VIII:C epitopes. One epitope on the C-terminal, 80,000 dalton fragment of VIII:C was identified with cross-reaction among 3 MoAbs (Synbiotics, Chemicon, and IB3). Another unique site on this same fragment was recognized with C7F7. Two MoAbs (RFF-VIIIC/6 and RFF-VIIIC/8) defined another site with cross-reactive idiotypes on the N-terminal, 90,000 dalton fragment. Hybritech MoAb identified a fourth functionally distinct site to which no other cross-reacting MoAbs bound. A fifth functional locus was defined with RFF-VIIIC/2 which reacted with an N-terminal site (distinct from the RFF-VIIIC/6 X RFF-VIIIC/8-defined site). A sixth functional locus was recognized with RFF-VIIIC/5 which reacted with a C-terminal site (distinct from the Synbiotics/Chemicon/IB3-defined site but possibly near the C7F7-defined site). RFF-VIIIC/10 identified a non-functional locus on the middle region of VIII:C. These MoAb-based assays resolve six sites to which high-titered inhibitors are directed and offer a path to further study the immunoregulation of human anti-VIII:C inhibitors.

Comparative behaviour of the components of the factor VIII complex in acute myocardial infarction.

The three components of the factor VIII complex: VIII coagulant (VIII:C), VIII related antigen (VIII R:Ag) and the VIII related von Willebrand factor (VIII R:WF) were studied in patients with acute myocardial infarction (AMI). Using a carefully standardized technique for the determination of VIII R:WF, a significantly higher R:WF level was found in 32 patients compared to 19 control subjects, confirming our previous results. However VIII R:AG was increased to an even greater extent, resulting in a VIII R:Ag/VIII R:WF ratio of 1.58 +/- 0.084 in patients, compared to 1.21 +/- 0.045 in controls. A similar increase of the VIII R:AG/VIII:C ratio was noted in the 13 patients in whom VIII:C was investigated. In 7 patients with severe AMI who could be investigated twice the plasma levels of both VIII R:Ag and VIII R:WF were found to be lower a week after the acute event than during the first 48 hours. However the VIII R:Ag/VIII R:WF ratio was not significantly reduced after 7 days. Acute phase reaction and endothelial injury resulting in release of multimers which are less polymerised are probably involved in the above changes.