Partial Thromboplastin Reagents Research Articles

The multiple faces of the partial thromboplastin time APTT

Converting the PTT into the APTTIn his historical sketch The partial thromboplastin time:defining an era in coagulation,G.C.WhiteII[1]describesthegenesis in 1953 of the original partial thromboplastin time(PTT) [2].In 1961 animprovedversionwas publishedfrom mylaboratory [3] in which adding kaolin powder to a partialthromboplastin reagent optimally activated Hageman factor(HF) and, consequently, plasma thromboplastin antecedent[PTA, factor (F)XI] during the test’s incubation period. Todaycommercial reagents provide this function. Although stillcommonly referred to as the PTT, the correct name of theimproved test is the APTT (activated partial thromboplastintime). My memory of the events of long ago leading toconversion of the PTT into the APTT follows.In1956Ireportedthatafour-partoriginalPTTassaysystemcontainingeitheradsorbedoxplasma(sourceoffactorVIII)oraged serum (source of factor IX) as a correcting agent hadproved its worth as a simple sensitive screening test. It hadenabledmetodeterminethekindofhemophiliapresentineachof a previously unscreened large group of 162 hemophilicchildren and adolescents in Los Angeles [4]. By 1957 using thisassay system I had also identified three adults with plasmathromboplastin antecedent (PTA, FXI) deficiency.Thencameaproblem.Normal silicone testplasmafailedtoshorten the prolonged PTT of glass-activated substrate plasmafrom these three patients that had stood for 4 h at 4 Cbeforetesting.Incontrast,thesame silicone testplasmareadilydidsoif shaken with glass beads. Submitting these data to the JLabClin Med in January 1958, I postulated that plasma PTAcirculates as an inert precursor and emphasized the need tostandardize exposure to glass in constructing a quantitativeassay for PTA activity [5].In accepting my paper the editor, C. Loosli, informed me ofan abstract from the Proceedings of the Thirtieth AnnualMeeting of the Central Society for Clinical Research thatappeared in the December 1957 issue of the JClinLabMed.Itwas from Oscar Ratnoff and Jerold Rosenblum on the role inthe initiation of clotting by glass of HF [6]. A full paper fromRatnoff and Rosenblum was published in 1958 [7] and soonotherinvestigatorsconfirmedthatbothHFandPTA(FXI)arerequired for the glass activation of normal plasma.Bjarne Waaler’s 1959 monograph [8] of work carried outbetween 1955 and 1958 in Paul Owren’s Institute for Throm-bosis Research in Norway particularly interested me. Inaddition to confirming that both HF and PTA were requiredfor plasma to form an activation product after contact withglass, Waaler had found a cause for the disconcertingly widenormal range of the PTT test. Clotting times depended uponwhether new or previously used glass test tubes were used, andif the latter, how the tube had been washed. New glass tubesyielded50–60-sclottingtimeswhereasglasstubesthathadbeenwashed with a commonly used acid cleaning solution yielded80–90-s clotting times.Waaler also reported that the clotting time of plasmalengthened markedly if after exposure to glass powder, oneremoved the powder and allowed the plasma to stand forseveral hours at 37 C. He called such plasma exhaustedplasma and believed it was depleted of both HF and PTA. Iused his method to create an exhausted plasma substrate foruse in my laboratory.In late 1958 I was recruited as the full-time hematologist forthe Department of Medicine of the University of SouthernCalifornia (USC) Medical School and its clinical services at theLos Angeles (LA) County Hospital. By the fall of 1989 mytransplanted Coagulation Research Laboratory was up andrunning at USC. A young couple, Norman and SandraSchiffman, had moved to Los Angeles, he to enter medicalschool at USC and she to enter the PhD program of itsDepartment of Biochemistry. Arnold G. Ware of the Depart-ment of Biochemisty, who was the Director of the ClinicalChemistry Laboratory of the LA County Hospital, suggestedthatS. Schiffman,whilesearching fora thesis project, talk withme. Fortune smiled upon me. This wonderfully bright,energetic graduate student elected to work on a project in mylaboratory. She would separate PTA (FXI) and HF fromnormal plasma by ion-exchange chromatography using therecently available ion exchange resin diethylaminoethyl cellu-lose (DAEA).Although I had PTA-deficient, HF-deficient and exhaustedplasmas ready for use as substrates for assays of eluatefractions, I knew from the experience cited above that suchassays should not be based upon the original PTT procedure.J.Margolishadpublisheda paperin1958onanewtestthathecalled the kaolin clotting time [9]. When S. Schiffman and Iread his paper, for us, a light turned on. We would design fourcomponent assays for her project that contained equal parts ofa cephalin (partial thromobplastin)–kaolin (HF activator)suspension, substrate plasma, an eluate sample, and CaCl

Alpha 2-Macroglobulin remains as important as antithrombin III for thrombin regulation in cord plasma in the presence of endothelial cell surfaces.

Infants and children rarely develop thrombotic complications compared with adults, suggesting that there are protective mechanisms in place for the young. Because endothelial cell surfaces regulate thrombin formation and inhibition, we compared thrombin regulation by human umbilical vein endothelial cell surfaces exposed to defibrinated cord and adult plasmas. After activation by either 10% activated partial thromboplastin reagent (strong activator) or coagulant phospholipids (weak activator) the following were measured: free thrombin, thrombin bound to antithrombin III (ATIII), heparin cofactor II, alpha 2-macroglobulin (alpha 2M), and prothrombin concentration. Free thrombin activity was expressed as remaining activity, after subtraction of thrombin-alpha 2M activity. After 10% activated partial thromboplastin reagent, 100% of prothrombin was consumed and significant amounts of thrombin generated by 2 min. Cord plasma generated significantly less thrombin than adult plasma, reflecting the lower initial plasma concentration of prothrombin. correspondingly, concentrations of thrombin inhibitor complexes were significantly greater in adult plasma than in cord plasma. After coagulant phospholipids, 50% of prothrombin was consumed and negligible thrombin activity measured for both adult and cord plasma. Similar amounts of thrombin inhibitor complexes were formed. ATIII was the predominant inhibitor of thrombin in adult plasma, whereas alpha 2M was as important as ATIII in cord plasma for both activators. When cord plasma concentrations of ATIII were increased to adult values, the proportion complexed to alpha 2M decreased. We conclude that on human umbilical vein endothelial cells, the capacity to generate thrombin is decreased in adult and cord plasmas.(ABSTRACT TRUNCATED AT 250 WORDS)

The preparation of a sensitive partial thromboplastin reagent from bovine brain.

Commercial partial thromboplastin reagents markedly differ in their sensitivity to factor deficiency, heparin, or the lupus anticoagulant. These differences may be partly due to the variable phospholipid content of different commercially available reagents. For over 15 years, we have routinely used a partial thromboplastin prepared from human brain. In the past four years, we have been using a similarly prepared bovine partial thromboplastin reagent. This report describes the preparation of our partial thromboplastin reagent, as well as an analysis of the phospholipid composition of both the human and bovine thromboplastin reagents. Four separate brain preparations produced consistent percentages of the anionic phospholipids, phosphatidylserine, and phosphatidylinositol. The bovine reagent was also compared with commercial partial thromboplastin reagents in the detection of coagulation factor deficiency, heparin, and the lupus anticoagulant.

Trials of Commercial Reagents in Anion-exchange Coagulation Procedures: Ortho Diagnostics

The suitability of Ortho Diagnostics one-stage prothrombin time (PT) reagent (Ortho Brain Thromboplastin) and activated partial thromboplastin (aPTT) reagent (Activated Thrombofax) has been evaluated for use in conjunction with the anion-exchange heparin removal maneuver. The PT/HR and aPTT/HR are tests used to follow the anticoagulant influence of coumarins when heparin also is being administered. After establishing a coumarin therapeutic range for Activated Thrombofax, a parallel trial was conducted with Ortho Brain Thromboplastin on coumarin-treated patient plasmas. Determinations also were made after heparin (0.2 mu/mL) was added and then removed by ECTEOLA microchromatography columns. Ortho Brain Thromboplastin was found to induce a shortening bias associated with a spurious improvement in the precision of tests run on anion-exchange treated plasmas that potentially could result in coumarin overdosage. The systematic error did not appear to result either from protracted incubation or the activation of prekallikrein, high molecular weight kininogen, Factor XI or XII. This reagent was found to perform appropriately with plasma not exposed to ECTEOLA. Activated Thrombofax gave reliable and reproducible results before and after heparin removal. This aPTT reagent could be used in the aPTT/HR anticoagulant surveillance scheme.

Procoagulant activity of partial thromboplastin (cephalin) reagent and its phospholipid composition

The relationship between the phospholipid composition of successive batches of the standardised partial thromboplastin reagents produced in the National (UK) Reference Laboratory for Anticoagulant Reagents and Control and their procoagulant activity in the kaolin-activated partial thromboplastin time test (APTT) has been investigated. Separation and quantitation of individual lipid classes was achieved by the use of a modified high pressure liquid chromatographic technique to provide more rapid and improved separation of the lipid classes. Many batches of human brain partial thromboplastin reagent, of comparable activity in the standardised APTT technique, were investigated. Considerable variation was found in total lipid concentration of partial thromboplastin extracts with identical procoagulant properties. The coefficient of variation of individual components showed that phosphatidyl serine was the most consistently represented class amongst reagents studied. Phosphatidyl serine inhibited or promoted clotting activity in the APTT according to its concentration.

Whole-blood Clotting Time, Activated Partial Thromboplastin Time, and Whole-blood Recalcification Time as Heparin Monitoring Tests

The authors performed whole-blood clotting time (WBCT), activated partial thromboplastin time (APTT), and whole-blood recalcification time (WBRCT) tests on normal blood or citrated plasma, each milliliter containing 0-0.5 unit heparin, and on samples from patients, of whom many were receiving heparin anticoagulation therapy. Six partial thromboplastin reagents were used. Linearity between clotting time and heparin concentration was observed with WBCT and APTT, determined with Hyland partial thromboplastin (kaolin-activated) and Dade ("Improved" Activated Cephaloplastin and Actin) reagents. With a General Diagnostics preparation (Platelin -plus, celite as the activator) and another Hyland partial thromboplastin reagent (silica-activated), the sensitivity to heparin decreased to beyond 0.3 unit/ml plasma. No correlation was observed with the old Dade Activated Cephaloplastin reagent, WBRCT was completely insensitive to heparin in concentrations as high as 0.24 unit/ml blood. With patient samples, correlations were observed between WBCT and Hyland (kaolin) APTT, and between Hyland and Dade Actin APTT. However, WBCT and WBRCT, and APTT and WBRCT, correlated poorly.

Comparative Evaluation of a Partial Thromboplastin Reagent Containing a Non-settling, Particulate Activator

Five commercial reagents for determining the activated partial thromboplastin time were compared on an instrument with photometric clot detection. The activating agents employed in these reagents are ellagic acid, celite, kaolin and a new agent, colloidal silica. The reagent containing colloidal silica was the most sensitive to low levels of heparin. It also was the only reagent with substantial buffering capacity, and in precision on repetitive tests it was equal to or better than any of the other reagents evaluated. Although there were no striking differences among the reagents in sensitivities to moderate deficiencies in individual coagulation factors, the colloidal silica reagent was most sensitive to factor V and the reagents containing ellagic acid were significantly less sensitive to factors X and XII.

Studies on hypo- and hypercoagulability. II. Coagulation and fibrin analyses in severe infectious and toxic conditions.

Plasma phospholipids were elevated in patients with severe infections with renal complications (except for pyelonephritis) without shock in comparison with normals and with patients with severe infectionswithout renal complications. In contrast, in patients with toxic conditions with and without shock, total phospholipids were decreased. The percentages of phosphatidyl-ethanolamine and of lecithin were increased and those of lysolecithin decreased in all patient groups. By incubation of citrate plasma, partial thromboplastin or thromboplastin and Ca++ fibrins were produced, washed out, homogenized and phospholipids were estimated. In the patient groups with infections with renal complications and with peritoneal toxic conditions total phospholipids in the fibrins produced with partial thromboplastin reagent were significantly increased both in comparison to normals and to the corresponding amounts of reagent. Phospholipids in thromboplastinproduced fibrins were significantly elevated only in the patients with infections with renal complications. The results suport the view that plasma phospholipids-and herewith lipoproteins-are not simply trapped by the fibrin network, but play a role in the coagulation process, particularly under pathological conditions: Routine coagulation parameters (fibrinogen, one-stage prothrombin time, partial thromboplastin time, platelets, thrombelastography, factors II, V, VII, VIII, IX, X) varied greatly. PT* and factor II and VII were significantly decreased in patients with toxic conditions. Fibrinogen was augmented, but the platelet count decreased in patients with infections with renal complications. Of all parameters tested, the increases of percentages of phosphatidylethanolamine in plasma and of total phospholipids in PTT-produced fibrins were most closely connected with clinical signs of hypercoagulability (i.e. of intravascular coagulation without excessive consumption of clotting factors leading to bleeding).

Letter: Tissue thromboplastin and activated partial thromboplastin reagents.

Letter: Tissue thromboplastin and activated partial thromboplastin reagents.

The Use of a Stabilized Partial Thromboplastin Reagent in the Partial Thromboplastin Test

Abstract A stable sensitive partial thromboplastin reagent is prepared from an ether extract of acetone-dried bovine brain. The normal oxalated plasma partialthromboplastin time range with this reagent is 66-86 sec. Lyophilized plasmas deficient in coagulation factors give prolonged partial thromboplastin test times. Some of the properties of this reagent are discussed.