Postoperative stroke in a child with cerebral palsy heterozygous for factor V Leiden.

A 5-year-old with spastic quadraparetic cerebral palsy suffered multiple strokes after extensive orthopedic surgery. Coagulation testing was undertaken to determine whether a familial thrombophilia was present. The patient was found to be heterozygous for factor V Leiden. Factor V Leiden may be a risk factor for central nervous system events in special-needs children, particularly when common medical conditions create additional procoagulant risks.

Detection of the factor VLeiden mutation. Development of a testing algorithm combining a coagulation assay and molecular diagnosis.

The Coagulation and Molecular Diagnostic laboratories at the University of Minnesota Medical School (Minneapolis) have collaborated to develop a diagnostic algorithm to identify all factor VLeiden mutation carriers without performing unnecessary and expensive genetic testing. The algorithm uses a coagulation assay for activated protein C resistance (APCR) to determine the need for genetic testing. We report the results of our experience validating this program. We compared the sensitivity, specificity, and positive and negative predictive values of two measures of APCR, the APCR ratio and the normalized ratio. We found that the normalized ratio was the more sensitive but less specific parameter to determine the need for genetic testing. By using the normalized ratio as the standard by which to refer patients to the Molecular Diagnostics Laboratory, all mutation carriers were identified. We found a large overlap in both measures of APCR between symptomatic patients with normal genotype and mutation carriers. Furthermore, we demonstrated that increased factor VIII levels with a normal genotype are associated with apparent APCR. In this article we also review other correlates of apparent APCR.

Laboratory diagnosis of inherited protein S deficiency.

The authors investigated assays for free protein S (ProS) antigen, total ProS antigen, and ProS crossed immunoelectrophoresis (CIEP) in the diagnosis of type 1 inherited ProS deficiency. Accurate measurement of the hemostatically important free ProS required showing that, on each specimen, precipitation of the C4b-binding protein (C4b-BP)/protein S complex (C4b-BP/ProS) by polyethylene glycol-8,000 (PEG) was complete. The authors showed this by doing a ProS CIEP on the same PEG supernate that was used for quantitative measurement of free ProS. The +/- 2 standard deviation (+/- 2 SD) ranges for free ProS were 81-133% for males and 50-130% for females. This striking male-female difference has been reported only twice before. With the use of a graph of values for free ProS versus prothrombin time (PT), patients with inherited ProS deficiency segregated cleanly from normals and from patients on warfarin therapy without ProS deficiency until the PT was greater than 20 seconds. There is overlap of total ProS antigen values between normals and patients with inherited ProS deficiency.

Absence of HIV-1 infection in antibody-negative sexual partners of HIV-1 infected hemophiliacs.

In order to confirm the presence and determine the frequency of human immunodeficiency virus, type 1 (HIV-1) infection prior to antibody production, 23 healthy women with histories of repeated unprotected sexual exposure to HIV-1 infected hemophiliacs were tested for evidence of HIV-1 infection. Female subjects were tested for HIV-1 antibody (enzyme immunoassay [EIA] and Western blot), HIV-1 serum antigen, HIV-1 DNA gag sequences by the polymerase chain reaction, and HIV-1 virus isolation from peripheral mononuclear cells. Twenty-two of 23 (96%) women were negative by all HIV-1 assays. One woman was positive by all the HIV-1 assays including an EIA screening test for HIV-1 antibody. These preliminary results suggest that the frequency of HIV-1 infection in antibody-negative sexual partners of HIV-1 infected individuals is probably very low.

Hemophiliacs with HIV antibody are actively infected.

Cultures of peripheral blood mononuclear cells for human immunodeficiency virus type 1 (HIV-1) and assays for the p24 antigen were performed for a group of 75 unselected hemophiliacs to determine whether patients positive for HIV-1 antibody are actively infected rather than immunized by viral proteins in non-heat-treated factor VIII or IX concentrates. Fifty-six (75%) of the 75 hemophiliacs were antibody positive and 55 (98%) of the 56 with antibodies also had positive cultures. The one culture-negative individual had detectable HIV-1 proviral DNA sequences in three separate samples of peripheral blood mononuclear cell DNA, as detected by a polymerase chain reaction assay. Detection of serum p24 antigen and the time to development of a positive culture were significantly more frequent and shorter, respectively, in symptomatic vs asymptomatic patients. None of the 19 hemophiliacs negative for HIV-1 antibody had positive cultures, detectable p24 serum antigen, or symptoms of HIV-1 infection. Moreover, latent HIV-1 infection was not detected in 16 female sexual partners of hemophiliacs positive for HIV-1 antibody using Western blot testing, assays for p24 antigen, HIV-1 cultures, and polymerase chain reaction assays, despite repeated unprotected sexual exposure. We conclude that antibody-positive hemophiliacs have been actively infected by HIV-1 and that a long period of latent HIV-1 infection prior to overt seroconversion is unlikely.

An insertion within the factor IX gene: hemophilia BEl Salvador.

A patient with moderate to severe hemophilia B has been found to have a large insertion within his factor IX gene. The site of insertion is located in a DNA segment of approximately 0.8 kb between exon IV and an EcoRI site within intron D. The size of the DNA insertion is approximately 6 kb, and it contains at least two TaqI sites, two EcoRI sites, and one HindIII site. The insert probably originates from outside the FIX gene and does not represent an internal duplication. We propose that this abnormal FIX gene be called FIX El Salvador in recognition of the birthplace of the patient.

Prevalence of cytomegalovirus antibody in hemophiliacs and homosexuals infected with human immunodeficiency virus type 1.

We determined the prevalence of antibody to cytomegalovirus (CMV) in the sera of non-homosexual hemophilia patients and homosexual men infected with the human immunodeficiency virus type 1 (HIV-1). CMV antibody testing by latex agglutination revealed 33 of 58 HIV-1 infected hemophiliacs (57%) were antibody-positive compared with 54 of 54 HIV-1 infected asymptomatic non-hemophiliac homosexuals (100%) (p less than .001). Nine of 15 hemophiliacs (60%) with symptomatic HIV-1 infection were CMV antibody-positive. We also tested 22 HIV-1 antibody-negative hemophiliacs who had received non-heat treated factor concentrates. 14 of these 22 (64%) were CMV antibody-positive compared with 57% of HIV-1 antibody-positive hemophiliacs. We conclude 1) there is little correlation between transmission of HIV-1 and CMV by factor concentrates, 2) the presence of CMV antibody does not appear to be associated with clinical stage of HIV-1 infection in hemophiliacs, and 3) there may be a significant number of CMV antibody-negative hemophiliacs with HIV-1 infection at risk for primary infection and subsequent disease if CMV seronegative blood products are not provided for future transfusions.

Heparin cofactor II assay. Elimination of heparin and antithrombin-III effects.

Functional assays for heparin cofactor II (HC-II) are based on the inactivation of thrombin by HC-II in the presence of dermatan sulfate (DS). Residual thrombin is measured in a chromogenic assay. Interference by the antithrombin-III (AT-III)/heparin complex, which also rapidly inactivates thrombin, must be eliminated from the HC-II test system. Commercial DS is contaminated with heparin, while plasma specimens to be tested contain AT-III. After NaNO2/acetic acid treatment of DS (to inactivate heparin), there was enough residual heparin to cause AT-III interference. Treatment of plasma with commercially available anti-AT-III antiserum largely, but not completely, removed AT-III interference from the HC-II assay. With commercially available reagents, both NaNO2/acetic acid treatment of DS and anti-AT-III treatment of plasma were needed to eliminate heparin/AT-III interference. Protamine sulfate inactivated DS as well as heparin and could not be used to reduce AT-III/heparin interference with the HC-II assay.

Kallikrein inhibition and C1-esterase inhibitor levels in patients with the lupus inhibitor.

It has been suggested that kallikrein inhibition may predispose patients with the lupus inhibitor to thrombosis by interfering with the Factor XII-mediated activation of plasminogen. To further investigate this suggestion, the authors measured kallikrein inhibition in 19 patients with the lupus inhibitor. They found that kallikrein inhibition was greater than 100% of that of a normal plasma pool in all patients and greater than 125% in 11 of 19. Kallikrein inhibition was significantly correlated with C1-esterase inhibitor (C1S-INH) concentration, which they measured by rocket immunoelectrophoresis (r = +0.55, P less than 0.05). In three patients the C1S-INH was more than 30% greater than the kallikrein inhibition. Crossed immunoelectrophoresis for C1S-INH in these patients' plasma revealed an electrophoretic mobility identical with that of the normal plasma pool. The authors suggest that C1S-INH-mediated kallikrein inhibition, in conjunction with other coagulation abnormalities, predisposes patients with the lupus inhibitor to thrombosis.

A human myeloma-produced monoclonal protein directed against the active subpopulation of von Willebrand factor.

The authors present a study of a human myeloma-produced monoclonal protein (IgG-k) directed against von Willebrand factor that caused an acquired von Willebrand's disease (vWD)-like syndrome. The illness was characterized by upper gastrointestinal bleeding, prolonged bleeding time, decreased platelet adhesiveness, lack of platelet aggregation in response to ristocetin, and a qualitatively abnormal Factor VIII related antigen (vWF) by two-dimensional immunoelectropheresis. Patient plasma or IgG fraction mixed with normal platelet-rich plasma completely inhibited aggregation with ristocetin, but patient platelets resuspended in normal plasma aggregated normally with ristocetin. VWF was markedly elevated and the two-dimensional immunoelectropheresis of vWF revealed a vWD type II-like pattern with an absence of the higher molecular weight forms of the vWF. Marked inhibitory activity was observed in the ristocetin cofactor assay but disappeared at the highest dilutions of patient plasma used in the assay. Infusion of cryoprecipitate following plasmapheresis led to a correction of the bleeding time, improvement in platelet adhesiveness, transient disappearance of inhibitory activity in the Factor VIII ristocetin cofactor assay, and no significant normalization of two-dimensional immunoelectropheresis of vWF. This case demonstrated a myeloma-associated monoclonal antibody that interacted specifically with that part of the Factor VIII molecule necessary for Factor VIII ristocetin cofactor activity, normal platelet adhesiveness, and bleeding time.

Abnormal prothrombin crossed-immunoelectrophoresis in patients with lupus inhibitors.

Prothrombin deficiency has been known to occur in association with lupus inhibitors for over 25 years. We studied 21 patients with lupus inhibitors and found that four of five with prothrombin deficiency and ten of 16 with quantitatively normal prothrombin had abnormal prothrombin crossed-immunoelectrophoresis (CIEP) characterized by material moving slower in the first dimension of electrophoresis than normal prothrombin. In two patients with prothrombin deficiency, all prothrombin measured by quantitative assay and all slow-moving material on CIEP were removed by treatment with Staphylococcal protein A (SPA). These patients had free antibody, which bound to normal plasma prothrombin, forming larger amounts of slow-moving material on CIEP. A third patient with prothrombin deficiency had only partial removal of prothrombin after SPA treatment. Two patients with quantitatively normal prothrombin had all slow-moving material on CIEP and about one fourth of the prothrombin by quantitative assay removed by SPA treatment. There was no correlation among the strength of the inhibitor, the presence of a "cofactor effect," and the prothrombin abnormality. These data suggest that heterogeneous antiprothrombin antibodies, with or without prothrombin deficiency, are present in the majority of patients with lupus inhibitors.

Tympanostomy tubes in children with hemophilia A.

With proper teamwork between the hemotologist and otolaryngologist, children with hemophilia A and otitis media can obtain the benefits of tympanostomy tubes; 90% phenol applied topically to the tympanic membrane is a useful hemostatic agent. The technique may allow the hemophiliac child to undergo myringotomy and insertion of tympanostomy tubes without the use of blood products. There is evidence that the use of such blood products derived from pooled human plasma may be associated with the development of acquired immune deficiency syndrome (AIDS).

The effect of L-asparaginase on antithrombin, plasminogen, and plasma coagulation during therapy for acute lymphoblastic leukemia.

Hemostatic function was studied sequentially in 12 children receiving L-asparaginase, vincristine, and prednisone as remission induction chemotherapy for acute lymphoblastic leukemia. The three-week period of L-asparaginase therapy was characterized by progressive decreases in plasma antithrombin, plasminogen, and fibrinogen concentrations, and by progressive increases in plasma clotting times (prothrombin time, partial thromboplastin time, thrombin time). Platelet counts rose rapidly during the third and fourth weeks of therapy as bone marrow remission was achieved. Factor V levels increased steadily during a five-week period, perhaps related to vincristine or prednisone therapy. Recent reports of thrombosis and hemorrhage in children and adults receiving L-asparaginase may be explained by this complex set of abnormalities in coagulation and coagulation control.