Evaluation of four hematology and a chemistry portable benchtop analyzers using non-human primate blood.

BACKGROUND: Near patient testing (NPT) and point-of-care testing (POCT) using portable benchtop analyzers has become necessary in many areas of the medical community, including biocontainment. METHODS: We evaluated the Beckman AcT diff, Abaxis Vetscan HMII (two instruments), Abbott Cell-Dyn 1800, and Abaxis Vetscan VS2 for within-run precision and correlation to central laboratory instruments using non-human primates blood. RESULTS: Compared with the central laboratory instruments, the Beckman AcT diff correlated on 80%; the HMII instruments on 31% and 44%, the CD1800 on 31%, and the VS2 on 71% of assays. For assays with published manufacturers precision guidelines, the AcT diff met all nine, the HMII instruments met one and six of six, and the CD 1800 met one of six. CONCLUSIONS: Laboratories using NPT/POCT must test their individual instruments for precision and correlation, identify assays that are reliable, and exclude or develop supplemental procedures for assays that are not.

Drawing specimens for coagulation testing: is a second tube necessary?

Three recent studies discussed the possibility that the National Committee for Clinical Laboratory Standards (NCCLS) recommendations that the coagulation specimen should be the second or third tube collected are unnecessary. However, only one reagent/instrument was used in each study. Our protocol differed from the previous studies because we performed the assays on three different reagent/instrument systems on the same samples. Our study used photo-optic, mechanical, and nephelometric systems of clot detection. After obtaining informed consent, we obtained two blue-stoppered tubes of blood from 95 subjects: 15 normal patients and 80 patients currently on coumadin therapy. No discard tube was drawn for coagulation testing. A prothrombin time with an international normalized ratio and an activated partial thromboplastin time, were performed on each tube. Laboratory One used a MLA 1600C (Hemoliance) with Thromboplastin DS (Pacific-Hemostasis, ISI of 1.11) and APTT-LS (Pacific-Hemostasis). Laboratory Two used an STA (Diagnostica-Stago) with Neoplastine CI+ (Diagnostica-Stago, ISI of 1.14) and PTT-LT (Diagnostica-Stago). Laboratory Three used an ACL 300 with Plastinex (Biodata, ISI of 1.67) and Actin FSL (Dade Behring). No clinical or statistically significant differences were seen between the first or second tubes on any of the three reagent/instrument combinations in the PT in seconds, international normalized ratio reporting, or APTT results. Our results indicate that the NCCLS guidelines for obtaining a second tube when performing coagulation testing should be considered for elimination when new revisions are published.

A review of variables affecting PTs/INRs.

The prothrombin time is a common method of monitoring patients undergoing oral anticoagulant therapy. The proliferation of commercial thromboplastin brands with different international sensitivity indices (ISI) in conjunction with wider availability of automated coagulation analyzers has elevated the need for standardization in monitoring therapy.

A comparison of human and killer whale platelet fatty acid composition.

Activation of blood platelets and their subsequent aggregation results from the interactions of several complex metabolic pathways. Considered to be of critical importance are the platelet lipids. Subsequent to platelet activation, several membrane lipids undergo hydrolysis and the free fatty acids are metabolized to prostanoids which mediate platelet function in response to vascular injury. It is conceivable then, that differences in platelet membrane fatty acid content could result in significant differences in platelet responses to aggregatory stimuli, especially between species. The objective of this study was to identify specific differences in fatty acid content between human and killer whale platelets. Blood was collected, washed platelets were prepared, and platelet fatty acids were extracted. Methyl esters of the extracted fatty acids were analyzed by gas chromatography and reported as relative concentrations. Analysis of the data revealed significant differences between the two species for several relevant fatty acids, i.e. 16:0 (P < 0.05), and 18:0, 18:1, 18:2, and 20:4 (P < 0.001). The differences in platelet fatty acid composition and concentration may explain at least some of the differences in platelet function which have previously been identified between these species.

Oral anticoagulant therapy and international normalized ratios in swine.

While monitoring coagulation testing in Yucatan miniature swine being given oral anticoagulants, we noticed instances of high international normalized ratios (INR) without clinical complications in our animal model. All pigs (n = 17) weighed approximately 35.2 kg and were dosed daily with 2 to 3 mg of coumadin. Plasma samples were obtained and assayed for prothrombin time (PT), calculated INR, and activated partial thromboplastin time (APTT) at baseline, and after 7 and 14 days of coumadin therapy. Results of initial testing indicated high INR values after anticoagulation and short APTT values at baseline, which led us to consider the activity of vitamin K-dependent coagulation factors in the pig. This information was not available in literature concerning this strain of swine, and was surprising given that pigs are frequently used cardiac research models. Using the same plasma samples, we repeated the PT, INR, and APTT determinations using different reagents and a different analyzer. We also determined activities of coagulation factors II, VII, IX, and X. Large PT and INR differences were seen between the two instrument/reagent combinations, possibly due to the differences in the thromboplastins used and differences in the photo-optic versus manual clot-detection method of the instruments. Vitamin K-dependent factors in all pigs responded to coumadin by decreasing to < 30.0% activity, except for factor IX. The high INR values were not as pronounced when the second instrument/reagent combination was used, and the results seemed more in line with the animals' clinical condition. With this instrument/reagent combination, the pig can be considered a good model for research requiring oral anticoagulant medication.

Rapid removal of platelets from plasma utilizing the Hepcheck heparin removal filter.

We investigated whether Hepcheck heparin removal filters could remove residual platelets from platelet-poor plasma (PPP) without compromising samples for lupus anticoagulant (LA) testing. Furthermore we assessed what effect, if any, plasma filtration has on various clotting tests that form the foundation for LA testing. Citrated blood was obtained from 35 normal donors. Two sets of citrated tubes were processed in order to obtain PPP. Citrated blood was also obtained from a single donor to check the actual amounts of platelets removed by the Hepcheck filtration device. One set of PPP samples was filtered using the Hepchek filter device and the other was not processed, i.e. unfiltered. Prothrombin time (PT), activated partial thromboplastin time (APTT), and kaolin clotting time (KCT) were performed on both unfiltered and filtered samples that were tested immediately and after freezing at -70 degrees C for 24 h. Platelet counts on the single donor's citrated plasma were dramatically reduced after filtration. PT and APTT values showed small but statistically significant differences between unfiltered and filtered plasmas whether these were fresh or frozen samples. However, these differences were not clinically significant. KCT data showed statistical and clinical differences between unfiltered and filtered plasmas whether fresh or frozen plasmas were used. In contrast, KCT values were similar if unfiltered, fresh plasmas or filtered, frozen plasmas were used. Coagulation factor assays for factors VIII, IX and X were performed on both sets of PPP samples after freezing to determine if the filtration device affected these levels and would as a result, compromise APTT based lupus testing. Factor IX levels demonstrated a loss of activity following use of the device but no change was observed in factor VIII or factor X. Von Willebrand factor antigen and function as well as multimer structure were not affected by the filtration device in 10 normal donors. Filtering plasmas of two donors with a history of an LA dramatically prolonged clotting times for APTT, Dilute Viper Venom Time, mixing studies, and STACLOT LA tests in comparison with unfiltered plasmas. The data indicate that plasma filtration using the Hepchek device does not adversely affect coagulation testing. Furthermore samples requiring testing for the lupus anticoagulant can be filtered and subsequently frozen and compare favorably with freshly processed samples.

Effects of repeated freeze-thaw cycles on anticardiolipin antibody immunoreactivity.

The effect of repeated freeze-thaw cycles on anticardiolipin antibody levels was evaluated using an enzyme-linked immunosorbent assay. Normal human serum was spiked with known quantities of freeze-dried human polyclonal anticardiolipin antibody IgG and IgM (19 samples each) or IgA (11 samples). Each spiked sample was split into four identical aliquots; one aliquot was never frozen, and the remaining three were taken through successive freeze-thaw cycles. All aliquots from each sample were evaluated on the same day using the same plate and reagents. A significant decline in mean anticardiolipin IgG levels occurred between the aliquot which had never been frozen and the one which had been through three freeze-thaw cycles (Student's t-test, P = .04). Although mean IgM and IgA values declined as well, the differences were not significant. When individual samples were evaluated the decline appeared to occur most often between the second and third freeze-thaw cycle. Eight anticardiolipin IgG and three IgM-containing samples which had been positive initially became negative by the third freeze-thaw cycle. These data show that handling and storage of serum used to perform anticardiolipin antibody assays are important potential sources of assay variability.

Evaluation of a modified procedure for Staclot LA for the confirmation of lupus anticoagulants.

Staclot LA is a hexagonal (II) phase phospholipid clotting assay used to confirm the presence of lupus anticoagulants (LA). However, there have been complaints that the procedure contains several incubation steps requiring 15 min of operator time. The authors were able to shorten this procedure to a single 5 min incubation without affecting assay sensitivity. Both procedures were performed on 45 known lupus anticoagulant positive specimens, 25 normal donors, eleven plasmas from patients with known factor VIII or factor V inhibitors and ten other specimens submitted for lupus anticoagulant or anticardiolipin antibody testing but without complete testing to confirm the presence of LA prior to testing with Staclot LA. Excellent agreement was observed between the two procedures with concurrence in 87 of 91 specimens (95.6%). Each method detected 39 of 45 LA positive specimens giving a sensitivity of 86.7%. This modification shortens technologist time by two-thirds without compromising assay sensitivity, which will allow for automation on commonly used coagulation analysers.

Serum endotoxin, tumor necrosis factor, and interleukin-6 response to total hip arthroplasty.

The purpose of this study was to determine if endotoxin, tumor necrosis factor (TNF), and interleukin 6 (IL-6) increased in patients during or after total hip arthroplasty. Endotoxin is absorbed from the gut lumen and stimulates macrophages to make TNF. TNF is one of the stimuli for the increased production of IL-6. All three entities cause fever. If an increase takes place in any of the three, it could contribute to the commonly observed postop fever in total hip patients. Five serum samples were obtained from 12 patients: preoperatively; after the acetabular component was seated intraoperatively; in the recovery room; day 1 postoperatively; and day 3 postoperatively. IL-6 and TNF were measured using monoclonal antibody tests and endotoxin was measured with a chromogenic limulus amoebocyte lysate assay. A statistically significant increase in all three parameters was demonstrated. Endotoxin peaked intraoperatively or in the recovery room, TNF tended to peak in the recovery room, and the peak in IL-6 was more variable. Ten patients had a concomitant postoperative fever.

ARACHnase. An evaluation of a positive control for platelet neutralization procedure testing with seven commercial activated partial thromboplastin time reagents.

ARACHnase (Hemostasis Diagnostics International Co., Denver, CO) is a normal plasma that contains a venom extract from the brown recluse spider, Loxosceles reclusa, which mimics the presence of a lupus anticoagulant (LA). Seven activated partial thromboplastin time (APTT) reagents were used for platelet neutralization procedure (PNP) testing with ARACHnase: Automated APTT (Organon-Teknika, Durham, NC); Thrombofax and Thrombosil (Ortho, Raritan, NJ); Actin and Actin FSL (Dade, Aguado, PR); and Thromboscreen-Kontact and Thromboscreen-APTT LS (Pacific-Hemostasis, Ventura, CA). ARACHnase consistently displayed a positive PNP result of greater than 5 seconds correction of the initial baseline APTT. Thus, ARACHnase may provide a positive control for LA testing, regardless of the choice of APTT reagent and activator/phospholipid combination.

Aggregation of killer whale platelets.

The aggregation of blood platelets is a crucial step in normal hemostasis for all mammals. Circulating platelets are sensitive to a large variety of physiologic and non-physiologic stimulants, some of which are formed or exposed in conjunction with vascular damage or endothelial cell denudation. In addition, drastic pressure changes activate human platelets. Killer whale platelet function, on the other hand, is very intriguing since these animals do not seem to experience untoward platelet reactions during or after diving to great depths, nor do they experience abnormal bleeding associated with sub optimal platelet function. We examined this concept and determined that killer whale platelets, in response to ADP, PAF, and arachidonic acid, appeared to aggregate normally during the first 2-5 minutes after addition of the agonist, but had completely disaggregated at 10 minutes. Collagen- and A23187-induced aggregation appeared normal and complete within 10 minutes, while there was no response to epinephrine or ristocetin. Thromboxane production by killer whale platelets appears to be quantitatively similar to that produced by human platelets in response to ADP and PAF and exceeded that produced by human platelets when collagen was used as the agonist. In summary, this study reports a reduced platelet aggregation reaction in killer whales in response to several platelet agonists which does not appear to be related to the generation of thromboxane. This phenomenon may serve a protective role in these mammals by preventing thrombosis during diving and resurfacing.