Undiluted Blood Research Articles

Plant Sterols Cause Macrothrombocytopenia in a Mouse Model of Sitosterolemia

Mutations in either ABCG5 or ABCG8 cause sitosterolemia, an inborn error of metabolism characterized by high plasma plant sterol concentrations. Recently, macrothrombocytopenia was described in a number of sitosterolemia patients, linking hematological dysfunction to disturbed sterol metabolism. Here, we demonstrate that macrothrombocytopenia is an intrinsic feature of murine sitosterolemia. Abcg5-deficient (Abcg5(-/-)) mice showed a 68% reduction in platelet count, and platelets were enlarged compared with wild-type controls. Macrothrombocytopenia was not due to decreased numbers of megakaryocytes or their progenitors, but defective megakaryocyte development with deterioration of the demarcation membrane system was evident. Lethally irradiated wild-type mice transplanted with bone marrow from Abcg5(-/-) mice displayed normal platelets, whereas Abcg5(-/-) mice transplanted with wild-type bone marrow still showed macrothrombocytopenia. Treatment with the sterol absorption inhibitor ezetimibe rapidly reversed macrothrombocytopenia in Abcg5(-/-) mice concomitant with a strong decrease in plasma plant sterols. Thus, accumulation of plant sterols is responsible for development of macrothrombocytopenia in sitosterolemia, and blocking intestinal plant sterol absorption provides an effective means of treatment.

Fluorescent labeling, sensing, and differentiation of leukocytes from undiluted whole blood samples

Abstract In this paper, we demonstrated leukocyte labeling, sensing and differentiation from undiluted human whole blood samples with microfabricated devices. A challenging issue in leukocyte sensing from blood samples is the required high-dilution level, which is used mainly to prevent interference from the overwhelmingly outnumbered erythrocytes. Dilution is undesirable for micro hemacytometers. It not only increases sample volume and processing time, but also requires mixing and buffer storage for on-chip implementation. Unlike commercial bulk instruments and previous efforts by other groups, we completely eliminated the requirement for dilution by staining leukocytes specifically with fluorescent dye acridine orange (AO) in undiluted human whole blood and then sensing them in microfluidic devices. Green fluorescent signal centered at 525 nm is used for leukocyte count and red fluorescent signal centered at 650 nm is used for leukocyte differentiation. Throughput of 1000 leukocytes per second was achieved.

Reversible detection of proteases and their inhibitors by a pulsed chronopotentiometric polyion-sensitive electrode

Polymer membrane electrodes operated by pulsed chronopotentiometry have recently been introduced to replace traditional ion-selective electrodes for a number of applications. While ion-selective electrodes for the polycation protamine have been reported, for instance, a pulsed chronopotentiometric readout mode (called here pulstrode) provides improved stability and reproducibility while exhibiting sufficient selectivity for the direct detection of protamine in undiluted whole blood samples. Here, such protamine-sensitive pulstrodes are applied for the real-time detection of the activity of the protease trypsin and its soybean inhibitor. This is possible because small fragments produced by the trypsin digestion are not detectable by the protamine-sensing membrane. The real-time response to the proteolytic reaction is shown to exhibit good reproducibility and reversibility, and the initial reaction rate is dependent on the concentration of the protease and its inhibitor.

Direct detection of tuberculosis infection in blood serum using three optical label-free approaches

We describe a new and rapid method for diagnosing tuberculosis-specific antibodies in undiluted human blood serum by surface sensitive biosensors. An integrated optical grating coupler, an interferometric biosensor and a device based on the reflectometric interference spectroscopy (BIAffinity, Analytik Jena AG) were used to determine tuberculosis-specific antibodies, which are present in blood serum of infected persons. The specific sensor surface was generated by immobilized 38-kDa tuberculosis antigen and real-time detection was performed without any label in all cases. Unspecific binding effects caused by serum proteins were suppressed by appropriate coupling chemistry. 1,4-Phenylenediisothiocyanate was used as a crosslinker for the coupling of the antigen achieving the effect of complete surface coverage. This enables label-free serodiagnosis of tuberculosis infection with a specificity of 100% and a sensitivity of 75%. Based on our results, we anticipate that a label-free detection procedure with one or more marker antigens is possible for a rapid, economical and accurate routine diagnosis of tuberculosis and other infectious diseases in the future.

THIS ARTICLE HAS BEEN RETRACTED The impact of a medium molecular weight, low molar substitution hydroxyethyl starch dissolved in a physiologically balanced electrolyte solution on blood coagulation and platelet function in vitro

Background and Objectives Hydroxyethyl starches (HES) may have the potential to impact negatively on haemostasis. Recent findings suggest that side‐effects on haemostasis stem not only from the physicochemical differences between HES, but also from the composition of the solvent. We compared the effects of a newly developed medium molecular weight (MW) and low molar substitution (MS) HES dissolved in a physiologically balanced electrolyte solution (MW 130, MS 0·42; B‐HES) with a commercially available non‐balanced HES (MW 130, MS 0·4; NB‐HES), and with Ringer's lactate (RL) solution in vitro.Materials and Methods Activated partial thromboplastin time (APTT), factor VIII clotting activity (F VIII:C) and von Willebrand factor (vWF) activity were investigated in 48 healthy individuals. Platelet function as measured by turbidimetric platelet aggregometry and whole blood impedance aggregometry induced by adenosine diphosphate (ADP), collagen and thrombin receptor activating peptide (TRAP), and by ADP and TRAP‐induced expression of activated platelet fibrinogen receptor glycoprotein (GP) IIb/IIIa was determined in 24 participants. Haemodilution (25% and 50%, v/v for blood coagulation analyses and 20% and 40%, v/v for platelet function studies) was performed using the two HES preparations and RL.Results APTT was significantly longer and F VIII and vWF significantly lower at 25% and 50% dilutions with NB‐HES compared to B‐HES and RL. At 20% and 40% dilutions, ADP and TRAP‐induced expression of activated platelet surface GP IIb/IIIa was significantly increased by B‐HES compared to NB‐HES and RL. Percentages of platelet GP IIb/IIIa expression were also significantly greater in samples diluted with B‐HES than in undiluted blood. Neither the diluent (B‐HES, NB‐HES and RL) nor the degree of dilution (undiluted, 20% and 40% dilution) had any significant influence on ADP, collagen or TRAP‐induced turbidimetric platelet aggregation or impedance platelet aggregation.Conclusions In contrast to a non‐balanced 130 kDa, MS 0·4 HES (NB‐HES), a 130 kDa, MS 0·42 HES preparation dissolved in a physiologically balanced electrolyte solution (B‐HES) does not affect APTT, F VIII:C and vWF in vitro. Both types of HES do not affect platelet aggregation induced by ADP, collagen or TRAP. B‐HES but not NB‐HES increases the expression of activated platelet GP IIb/IIIa induced by ADP or TRAP.

Polymerized Nile Blue derivatives for plasticizer-free fluorescent ion optode microsphere sensors

Lipophilic H +-selective fluorophores such as Nile Blue derivatives are widely used in ISE-based pH sensors and bulk optodes, and are commonly dissolved in a plasticized matrix such as PVC. Unfortunately, leaching of the active sensing ingredients and plasticizer from the matrix dictates the lifetime of the sensors and hampers their applications in vivo, especially with miniaturized particle based sensors. We find that classical copolymerization of Nile Blue derivatives containing an acrylic side group gives rise to multiple reaction products with different spectral and H +-binding properties, making this approach unsuitable for the development of reliable sensor materials. This limitation was overcome by grafting Nile Blue to a self-plasticized poly( n-butyl acrylate) matrix via an urea or amide linkage between the Nile Blue base structure and the polymer. Optode leaching experiments into methanol confirmed the successful covalent attachment of the two chromoionophores to the polymer matrix. Both polymerized Nile Blue derivatives have satisfactory pH response and appropriate optical properties that are suitable for use in ion-selective electrodes and optodes. Plasticizer-free Na +-selective microsphere sensors using the polymerized chromoionophores were fabricated under mild conditions with an in-house sonic microparticle generator for the measurement of sodium activities at physiological pH. The measuring range for sodium was found as 10 −1–10 −4 M and 1–10 −3 M, for Nile Blue derivatives linked via urea and amide functionalities, respectively, at physiological pH. The observed ion-exchange constants of the plasticizer-free microsphere were log K exch = −5.6 and log K exch = −6.5 for the same two systems, respectively. Compared with earlier Na +-selective bulk optodes, the fabricated optical sensing microbeads reported here have agreeable selectivity patterns, reasonably fast response times, and more appropriate measuring ranges for determination of Na + activity at physiological pH in undiluted blood samples.

PRESERVING LYMPHOCYTE RIBONUCLEIC ACID FOR ANALYSIS AND TRANSPORT FROM WHOLE BLOOD IN RURAL AREAS.

Background Preservation of human ribonucleic acid (RNA) from whole blood samples in rural areas with very few resources has previously been very difficult and/or expensive. This has limited clinical research trials in third world countries and distant rural areas in developed countries. The major drawback to being able to study these populations is the difficulty in transporting blood samples while maintaining the integrity and preventing degradation of the lymphocytes, DNA, protein, and RNA. Of these, RNA is the most difficult and expensive to preserve. Objective To develop an inexpensive, simple, and reliable method to collect and preserve high-quality RNA from whole blood lymphocytes for prolonged transport. Methods Blood was collected from young adult rats and treated four different ways. For the first method, blood was placed in an equal volume of RNAlater® and frozen in dry ice and then placed in −80°C for 1 week. This was then thawed on ice and processed. The second method used an equal volume of RNAlater® but was not frozen. The third method was diluted with an equal volume of water before processing. In the fourth method, fresh, undiluted blood was used. RNA was isolated with Trizol® LS immediately after collection or thawing. cDNA was then synthesized from this RNA. An RT-PCR was then run using the housekeeping gene GAPDH. Results The sample that had the highest expression was the untreated and undiluted whole blood sample at an average of 22.57 Ct. The second highest expression was the blood that was treated with RNAlater® and frozen for 1 week. This expressed at an average of 25.03 or 2.5 cycles later. The next highest expression was the blood diluted with water. The Ct value for it was an average of 30.89 or 8 cycles after the untreated blood. The fourth sample expressed at an average of 32.92 or over 10 cycles beyond the untreated blood. Conclusion We have successfully developed a simple, inexpensive, and reliable method of collection and stabilization of high-quality RNA from whole blood lymphocytes for transport and later experimental use. This preservation of RNA, when compared with fresh blood treated with RNAlater® or diluted with water, is expressed almost as highly as untreated or undiluted blood. We speculate that this procedure will greatly enhance the ability to study lymphocyte RNA in third world and rural area populations.

Evidence for an Enhanced Fibrinolytic Capacity in Liver Cirrhosis Measured with a New Global Fibrinolysis Test in Whole Blood.

It is known that liver cirrhosis is associated with hyperfibrinolysis, which might contribute to bleeding problems of patients with this disease. However, hyperfibrinolysis has been assessed by using tests (such as euglobulin, plasma or diluted whole blood clot lysis assays), which do not reflect all changes that might occur in the fibrinolytic parameters in blood. A recent study even questioned the presence of a hyperfibrinolytic state in nonbleeding cirrhotic patients, since no evidence of increased fibrinolysis was observed with a new plasma-based clot lysis assay (Gastroenterology 2001; 121:131). Therefore we decided to re-investigate the fibrinolytic state of cirrhotic patients with a recently developed test for global fibrinolytic capacity (GFC) using undiluted whole blood. Non-anticoagulated blood was collected from 30 healthy controls and 75 patients with cirrhosis of varying severity (34 Child-Pugh A, 28 Child-Pugh B and 13 Child-Pugh C). The GFC was determined immediately after collection by clotting the blood, incubating the clots during 3 hours at 37 degrees and then measuring the generated fibrin degradation products. Various haemostatic parameters were determined in plasma samples by using functional activity assays. The median GFC (25th–75th percentile) increased from 1.7 (0.8–3.5) mg/l in the controls to 4.0 (1.6–13.0) mg/l in Child-Pugh A (n.s.), to 11.1 (2.3–31.9) mg/l in Child-Pugh B (P<0.001) and to 22.5 (3.1–89.6) mg/l in Child-Pugh C (P<0.01). Plasma samples of the cirrhotic patients showed significantly decreased levels of antithrombin, plasminogen, alpha-2-antiplasmin and thrombin-activatable fibrinolysis inhibitor (TAFI), significantly increased levels of tissue-type plasminogen activator (tPA) and no significant change in plasminogen activator inhibitor-1 (PAI-1). Spearman correlation tests in the whole group (controls plus patients) showed significant correlations between GFC and antithrombin (r=−0.44), plasminogen (r=−0.38), alpha-2-antiplasmin (r=−0.45), TAFI (r=−0.40), tPA (r=0.77) and PAI-1 (r=−0.51) (all p-values <0.0001). In conclusion, the new GFC test, which includes all components present in blood, indicates that the fibrinolytic capacity is strongly increased in patients with severe cirrhosis. The anti-fibrinolytic effects of decreased plasminogen levels in cirrhosis are apparently overruled by the pro-fibrinolytic effects of decreased alpha-2-antiplasmin and TAFI levels and particularly of increased tPA activity levels.

Biomonitoring of 37 Trace Elements in Human Blood and Plasma From Inhabitants of Northern Germany By ICP-MS

P-145 Abstract: The trace elements Ag, As, Au, B, Ba, Be, Bi, Cd, Ce, Co, Cs, Cu, Ga, Hf, Hg, In, La, Mn, Mo, Ni, Pb, Pd, Rb, Rh, Ru, Sb, Se, Sn, Sr, Te, Th, Tl, U, V, W, Y and Zr were determined in 130 human blood and plasma samples from occupationally non-exposed volunteers living in the greater area of Bremen in northern Germany. Information on exposure conditions of all human subjects were collected by questionnaire-based interviews, including smoking habits, seafood consumption and the type of dental alloys in the teeth. Statistical data (geometric mean values, selected percentiles, etc.) about the trace element concentrations are provided, intended to serve clinical chemists, toxicologists and epidemiologists planning and conducting research about exposition to metals or trace element deficiencies. The blood and plasma samples were analysed by inductively coupled plasma mass spectrometry (ICP-MS) with an octopole based collision/reaction cell. Relevant spectral interferences could be avoided by addition of a few mL/min of helium or hydrogen to the cell. Prior to introduction into the ICP the samples were diluted 1/10 (V/V) with a 0.1% Triton-X-100 and 0.5% (V/V) ammonia solution. For internal quality assurance we have analysed control materials from several suppliers in different concentration levels. External quality assurance was performed by participation in 3 national and international quality assessment schemes. The limits of quantification (LOQ) calculated to the undiluted blood range from 0.003 μg/L for U to 0.1 μg/L for Ga. The geometric mean concentrations of the 37 elements in blood range from the LOQs in the lower ng/L—range up to 2369 μg/L for Rb. Essential trace element concentrations in blood are in a small range for Mn (4.8–18 μg/L), Cu (720–1800 μg/L) and Se (85–182 μg/L). Other essential trace elements concentration ranges are 12–195 μg/L for B, 0.04–0.8 μg/L for Co and 0.06–4 μg/L for Mo. Geometric mean concentrations of some toxic elements, such as Pb, Cd and Hg in blood are 19, 0,4 and 0,8 μg/L, respectively. For other toxic elements (Be, Th, U) or elements without a known biological function (Hf, Ce, La, Te, Ga) many of the concentrations in the real samples are below the LOQs. Differences between blood and plasma concentration levels of trace elements are also discussed.

Monitoring of cyclosporine levels in transplant recipients using self‐administered fingerprick sampling

Use of C(2) monitoring for cyclosporine A (CsA) microemulsion results in improved clinical outcomes vs. trough (C(0)) monitoring. Logistical issues include accurate timing of the C(2) sample; requirement for sample dilution with most standard assay techniques; and inconvenience for patients. Recently, it has been shown that CsA concentrations in capillary blood correlate closely with levels in venepuncture samples, and that liquid chromatography tandem mass spectrometry (LC-MS/MS) can analyse CsA concentration using undiluted capillary blood from fingerprick samples. In a study to assess the feasibility of CsA monitoring, 52 stable heart transplant patients were provided with kits to take fingerprick trough and C(2) blood samples at home, returning them to the laboratory by post for LC-MS/MS analysis. In total, 225 samples were provided, of which 14 (6%) were unsuitable for analysis because of clotting (n = 10) or insufficient volume (n = 4). Discomfort was not a problem and initial difficulties that some patients reported in taking the samples resolved with experience. All samples were returned by the postal system in a timely manner. Use of fingerprick assays could allow transplant physicians to have access to C(2) levels when patients visit the clinic for review, and avoids the need for patients to attend the clinic or local healthcare centre solely for venepuncture. A barrier to more widespread introduction of fingerprick testing is likely to be lack of suitable MS facilities and trained personnel. In conclusion, self-administered fingerprick testing for CsA blood levels is practical to implement and highly convenient for patients and offers advantages for the transplant team.

Ethanol production by Candida albicans in postmortem human blood samples: Effects of blood glucose level and dilution

We present two cases in which the ethanol concentration in blood samples taken after death continued to increase in the absence of any remarkable increase in n-propanol concentration. Species of bacteria and yeasts, including Candida albicans were isolated from these samples. We then examined whether C. albicans, the most common yeast in the general environment, was able to produce ethanol in human blood stored at room temperature. Ethanol production increased as the glucose concentration increased, indicating that C. albicans produced ethanol from the glucose. Our results also suggested that C. albicans produced ethanol more easily in blood diluted by intravenous infusions that included glucose than in undiluted blood. These findings are useful for the evaluation of postmortem ethanol production in subjects whose blood has been diluted by infusions with glucose. Furthermore, there was no quantitative relationship between the amount of n-propanol detected and the amount of ethanol production: n-propanol appears to be an unreliable index of putrefaction and postmortem ethanol production by C. albicans. It is possible for the blood ethanol level to be high and n-propanol not to be detected, even if the subject has not been drinking alcohol. We reconfirmed the necessity of immediately adding sodium fluoride to samples for ethanol analysis to prevent postmortem ethanol production.

Recommendation for measuring and reporting chloride by ISEs in undiluted serum, plasma or blood: International Federation of Clinical Chemistry and Laboratory Medicine (IFCC): IFCC Scientific Division, Committee on Point of Care Testing and Working Group on Selective Electrodes

The proposed recommendation for measuring and reporting chloride in undiluted plasma or blood by ion-selective electrodes (ISEs) will provide results that are identical to chloride concentrations measured by coulometry for standardized normal plasma or blood samples. It is applicable to all current ISEs dedicated to chloride measurement in undiluted samples that meet the requirements. However, in samples with reduced water concentration, results by coulometry are lower than by ion-selective electrode due to volume displacement. The quantity measured by this standardized ISE procedure is called the ionized chloride concentration. It may be clinically more relevant than the chloride concentration as determined by coulometry, photometry or by ISE after dilution of the sample.

Determination of optical properties of human blood in the spectral range 250 to 1100 nm using Monte Carlo simulations with hematocrit-dependent effective scattering phase functions

The absorption coefficient mu(a), scattering coefficient mu(s), and anisotropy factor g of diluted and undiluted human blood (hematocrit 0.84 and 42.1%) are determined under flow conditions in the wavelength range 250 to 1100 nm, covering the absorption bands of hemoglobin. These values are obtained by high precision integrating sphere measurements in combination with an optimized inverse Monte Carlo simulation (IMCS). With a new algorithm, appropriate effective phase functions could be evaluated for both blood concentrations using the IMCS. The best results are obtained using the Reynolds-McCormick phase function with the variation factor alpha = 1.2 for hematocrit 0.84%, and alpha = 1.7 for hematocrit 42.1%. The obtained data are compared with the parameters given by the Mie theory. The use of IMCS in combination with selected appropriate effective phase functions make it possible to take into account the nonspherical shape of erythrocytes, the phenomenon of coupled absorption and scattering, and multiple scattering and interference phenomena. It is therefore possible for the first time to obtain reasonable results for the optical behavior of human blood, even at high hematocrit and in high hemoglobin absorption areas. Moreover, the limitations of the Mie theory describing the optical properties of blood can be shown.

Pathogen Control in Complex Fluids with Water‐coupled Excimer Lamps at 282 and 308 nm

Water-coupled excimer lamp systems have been developed to inactivate microorganisms within complex, low-optical quality, fluids. Monochromatic lamps were selected to minimize UV-B and UV-C absorption within the carrier fluids while maximizing deposition within specific chemical targets. Fundamentals, system scaling and power supply design are discussed. This work used two large-surface area excimer lamps as intense sources of near monochromatic radiation at 308 and 282 nm. Data are presented for two distinct fluid systems: flow-through processing of large-volume metalworking fluids used in heavy industry and batch irradiation of human blood plasma and platelet suspensions used in transfusion medicine. In the first, a 200-600 L/min reactor is used to control bacterial concentrations within metalworking fluids used in large-scale metal machining processes. Control is defined as the maintenance of 10(3) to 10(4) CFU/mL in fluids that without treatment would have concentrations over 10(7) CFU/mL. The second is a batch process for viral inactivation in undiluted blood bank products. Samples of fresh frozen plasma and platelet suspensions were spiked with high titers of porcine parvovirus (PPV) and irradiated at 308 and 282 nm. Although both wavelengths were effective at reducing PPV levels, 308 nm light resulted in both higher rates of viral inactivation (greater than 6 log units) and lower rates of fluid degradation.

Effects of protamine and heparin can be detected and easily differentiated by modified thrombelastography (Rotem®): an in vitro study

Precise coagulation monitoring might help prevent heparin-protamine mismatch and thus decrease postoperative blood loss. We therefore measured coagulation time (CT) by modified thrombelastography (Rotem) as a possible differential monitor of the effects of heparin and protamine. Undiluted and diluted blood samples from 26 healthy volunteers were spiked with increasing concentrations of heparin (0.1, 0.2, 0.4, 0.8 and 1 U ml(-1)). In addition, undiluted blood was spiked with protamine hydrochloride (0.1, 0.2, 0.4, 0.8 and 1.6 U ml(-1)), and we tested the effect of protamine on the reversal of heparin 0.4 U ml(-1). Heparin-containing samples were analysed using the heparin-sensitive INTEM test and the heparinase-containing HEPTEM test; protamine series were also analysed with the EXTEM test (tissue factor activation). CT by the INTEM test [CT-INTEM; median (min/max)] increased significantly and dose-dependently with increasing concentrations of heparin [control, 175 s (146/226); heparin, 1.0 U ml(-1) 1320 s (559/2100); P<0.001] and protamine [control, 172 s (150/255); protamine, 1.6 U ml(-1) 527 s (300/1345); P<0.0001]. Up to heparin concentrations of 0.4 U ml(-1), results were similar in undiluted and diluted blood samples. As expected, CT-HEPTEM remained within the normal range for all tested heparin concentrations (median 180-183 s), but increased similarly to CT-INTEM for increasing protamine concentrations. CT measurement using the Rotem technique appears to be a valuable tool for heparin-protamine management. For detection of heparin alone, protamine alone and the two combined, the ratio of CT-INTEM:CT-HEPTEM can be used to distinguish the effects of heparin excess (CT-INTEM:CT-HEPTEM>1) from those of protamine excess (CT-INTEM:CT-HEPTEM=1).

Voltammetric Detection of Heparin at Polarized Blood Plasma/1,2-Dichloroethane Interfaces

Heparin, a highly negatively charged polysaccharide, which has been used widely as an anticoagulant and antithrombotic, was detected by ion-transfer voltammetry at the interface between 1,2-dichloroethane and an aqueous buffer solution or undiluted blood plasma. Quaternary ammoniums with different numbers of methyl and long alkyl groups were examined as positively charged heparin ionophores using pipet electrodes filled with the organic electrolyte solutions of their tetrakis(4-chlorophenyl)borate salts. It was shown that octadecyltrimethylammonium most selectively facilitates interfacial heparin adsorption without interference from potential-dependent ionophore transfer into the aqueous phase. Water-filled pipet electrodes were also used to study the stoichiometry of the interfacial complex between a heparin molecule and multiple ionophore molecules, which is discussed as a counterion condensation effect. Stripping voltammetry based on facilitated heparin adsorption and desorption gives a detection limit of 0.012 unit/mL in 0.12 M NaCl buffered at pH 7.2, which is 1 order of magnitude lower than therapeutic heparin concentrations (>0.2 unit/mL) and is comparable to a detection limit of the most sensitive heparin sensor reported so far. The biomedical utility of ion-transfer voltammetry was demonstrated for the first time in an undiluted blood sample. Despite interferences by Na+, Cl-, and plasma proteins such as serum albumin, a detection limit of 0.13 unit/mL was obtained in sheep blood plasma with the stripping method.

Guidelines for sampling, measuring and reporting ionized magnesium in undiluted serum, plasma or blood: International Federation of Clinical Chemistry and Laboratory Medicine (IFCC): IFCC Scientific Division, Committee on Point of Care Testing

All analyzers with ion-selective electrodes for ionized magnesium (iMg) should yield comparable and unbiased results. The prerequisite to achieve this goal is to reach consensus on sampling, measurement and reporting. The recommended guidelines for sampling, measurement and reporting iMg in plasma ("plasma" refers to circulating plasma and the forms in which it is sampled: the plasma phase of anticoagulated whole blood, plasma separated from blood cells, or serum) or blood, referring to the substance concentration of iMg in the calibrants, will provide results for iMg that are approximately 3% greater than its true concentration, and 4% less than its true molality. Binding of magnesium to proteins and ligands in plasma and blood is pH-dependent. Therefore, pH should be simultaneously measured to allow adjustment of iMg concentration to pH 7.4. The substance concentration of iMg may be physiologically and consequently clinically more relevant than the substance concentration of total magnesium.

In vitro effects of local anaesthetics on the thromboelastographic profile of parturients

Post-dural puncture headache can be an incapacitating complication of obstetric epidural analgesia/anaesthesia and early or prophylactic epidural blood patch (EBP) is one of the treatment options. Although local anaesthetic (LA) agents have been shown to have anticoagulation effects in vitro, peri-partum women are known to be hypercoagulable. We postulated that the presence of residual LA might not result in impaired haemostasis of the EBP in parturients. Blood samples from 10 healthy term parturients were subjected to thromboelastography after the addition of four different LA (lidocaine, bupivacaine, levobupivacaine, and ropivacaine) preparations. There was a significant reduction in reaction (R) and coagulation (K) time (P<0.001, P<0.05) and an increase in alpha degrees angle (P<0.01) when comparing undiluted blood with the saline control group. Maximum amplitude (MA) and clot lysis (Ly30) did not change significantly despite the 50% dilution. The thromboelastographic parameters of all four LA-treated groups were no different from their saline controls and from each other. At clinical dosages, LA did not cause any hypocoagulable changes on the thromboelastographic profile of healthy parturients.

Complications of Hyperleukocytosis and Leukapheresis in Pediatric Acute Leukemias.

Leukapheresis is commonly used in children with acute leukemia and hyperleukocytosis (WBC >100 x 109/L). We analyzed the frequency of early complications of hyperleukocytosis in children with acute lymphoblastic leukemia(ALL) and acute myeloid leukemia(AML) and the frequency of complications attributed to leukapheresis. We included all children with acute leukemia and hyperleukocytosis, presenting to HSC between January 1992 and May 2002. ICH/stroke, pulmonary leukostasis, acute renal failure (ARF) and death were considered severe complications of hyperleukocytosis. 61 ALL and 8 AML were reviewed. All children received intravenous hyperhydration, urinary alkalinization and allopurinol. Of 61 ALL children, 16 (26.2%) underwent leukapheresis; median WBC count was 559 x 109/L (range 200–969). Median WBC of non-leukapheresed ALL children was 181 x 109 /L (range 101–392). Five (62.5%) of 8 AML children underwent leukapheresis; median WBC was 376 x 109/L (range 167–470). The remaining 3 had a median WBC of 146 x 109/L (range 114–230). 10/61(16.3%) ALL patients presented with severe complications within 6 to 8 hours of arrival; 9 were leukapheresed. ICH occurred in 2, one of whom failed to receive platelet transfusion at presentation because of a falsely high automated platelet count corrected after 12 hours by more senior staff. Six had respiratory distress thought to be related to pulmonary leukostasis; all but 1 improved after leukapheresis. Three developed ARF due to hyperuricemia pre-leukapheresis. While 2 received leukapheresis, all required dialysis. One early death occurred on day 22 due to enterococcal sepsis. 3/8 (37.5%) AML children developed complications related to hyperleukocytosis: 2 had pulmonary leukostasis, only 1 improved after leukapheresis; 1 died at 72 hours due to stroke. Three non-leukapheresed AML patients remained clinically well with hyperhydration and early chemotherapy. Leukapheresis reduced the WBC by an average of 40–45% in both ALL/AML. Procedural complications secondary to leukapheresis occurred in 18/21(85.7%) children. Femoral vein thrombosis occurred in 5 patients. Five became hypocalcemic; while only one was symptomatic, all were given calcium infusions. Other complications were: coagulopathy(n=6), sinus bradycardia(n=4), hypotension(n=4), hypertension(n=3), hypokalemia(n=4), and hypomagnesemia(n=1). The leukapheresis circuits of 5 children (3 ALL, 2 AML) were primed with undiluted packed RBC; 3 had new or progressive pulmonary leukostasis following leukapheresis, 1 of whom also developed a stroke. In summary, severe complications in children with hyperleukocytosis are common. Leukapheresis reduced WBC in most patients and appeared to improve pulmonary leukostasis in some. However, it did not prevent progression of ARF. Also, procedural-related complications are considerable. We have identified the following sources of potentially preventable morbidity: 1. Calcium infusions during the procedure should be used with extreme caution; 2. Circuits should not be primed with undiluted red blood cells as this may exacerbate hyperviscosity; and 3. Manual platelet counts must be performed in hyperleukocytosis. Furthermore, it is insufficient for these issues to be appreciated by senior staff only. Continuous education of inexperienced staff and leukapheresis team regarding the most appropriate use of this procedure should be emphasized. A randomized evaluation of the benefits versus risks of leukapheresis is needed.

Σ ESR

The aim of the study was to simplify the first Σ erythrocyte sedimentation rate (ESR) method (manual hematocrit adjustment to 0.35, sum of 4 sedimentation levels) and to confirm its clinical relevance. The erythrocyte sedimentation rate of undiluted blood samples from 576 patients was measured simultaneously with and without manual hematocrit adjustment to 0.35 to identify an approximate expression of the area under the curve and a formula for calculating the Σ ESR. The Σ ESR formula was based on the sum of 2 unadjusted sedimentation levels, at 30 and 60 minutes, together with the hematocrit value and the hemoglobin concentration. Σ ESR values in 274 healthy subjects showed a gaussian distribution, no difference between men and women, and no significant increase with age. In recent-onset arthritis or disk-related lumbosciatic syndrome, Σ ESR seemed to be a more reliable marker of inflammation than the Westergren ESR and C-reactive protein. We also obtained data clarifying the controversial relationship of ESR with lipid levels and arterial hypertension.