Accurate platelet counting in an insidious case of pseudothrombocytopenia.

Anticoagulant-induced aggregation of platelets leads to pseudothrombocytopenia. Blood cell counters generally trigger alarms to alert the user. We describe an insidious case of pseudothrombocytopenia, where the complete absence of Coulter counter alarms both in ethylenediaminetetraacetic acid blood and in citrate or acid citrate dextrose blood samples was compounded by the fact that the massive aggregates were exclusively found at the edges of the blood smear. Non-recognition of pseudothrombocytopenia can have serious diagnostic and therapeutic consequences. While the anti-aggregant mixture citrate-theophylline-adenosine-dipyridamole completely failed in preventing pseudothrombocytopenia, addition of iloprost to anticoagulants only partially prevented the aggregation. Only the prior addition of gentamicin to any anticoagulant used resulted in a complete prevention of pseudothrombocytopenia and enabled to count accurately the platelets.

Multicenter harmonization of common enzyme results by fresh patient-pool sera.

A region consisting of 19 clinical laboratories harmonized their calibration of seven common enzymes by using fresh patient-pool sera. One of the laboratories was chosen to act as Regional Reference Laboratory (RRL). This laboratory used internationally accepted (mostly IFCC) methods at 37 degrees C, with an intralaboratory CV < or = 2.5%. First, the reference ranges of the RRL were verified by analysis of a reference population and calculation of the results by a parametric method. Next, all laboratories, including the RRL, received six patient-pool sera and analyzed them at the same time on the same date. Enzyme calibration factors at each laboratory were converted on the basis of the slope, and occasionally the intercept, of regression analysis with the RRL and the individual laboratory. Before harmonization, the interlaboratory CVs varied from 16.9% to 61.6%. After harmonization, CVs decreased to between 5.0% and 9.5%. These results proved to be reproducible over a period of more than a year. Using internationally accepted inaccuracy and imprecision criteria, the achieved interlaboratory CVs permit the use of one set of reference ranges by all participating laboratories. Certified Reference Materials were analyzed, resulting in interlaboratory CVs as low as those achieved with patient-pool sera. These materials can act as commutable reference preparations, except for creatine kinase.

Screening for EDTA-dependent deviations in platelet counts and abnormalities in platelet distribution histograms in pseudothrombocytopenia.

Screening for pseudothrombocytopenia caused by in vitro platelet clumping has been performed in 45,000 subjects attending a general hospital. In our region, the observed prevalence of EDTA-induced pseudothrombocytopenia in blood samples with an initial platelet count below 150 x 10(9)/l was estimated to amount to 0.1%. EDTA-induced pseudothrombocytopenia was confirmed by detection of platelet aggregates by means of microscopic evaluation from the blood smear. In routine investigations, pseudothrombocytopenia could be highly suspected when the Sysmex NE 8000 showed characteristic peculiarities in the white blood cell (WBC) scattergram and histogram. Platelet aggregation is avoided in such cases by the use of citrate as an anticoagulant instead of EDTA. Pseudothrombocytopenia was detected in 46 subjects. As a screening test for pseudothrombocytopenia, increased cut-off values derived from the WBC histogram demonstrated 90% sensitivity and 100% specificity. Automated flagging for platelet clumps, deviations reflecting MPV, or PDW abnormalities revealed lower scores with respect to sensitivity.

Faecal nitrogen determination by near-infrared spectroscopy.

The well-known Kjeldahl method for the determination of faecal nitrogen is rather complex, time-consuming and expensive. Therefore, the use of near-infrared spectroscopy in determining the amount of nitrogen in faeces has been studied. To our knowledge we are the first to present the calibration equation for the determination of nitrogen with near-infrared spectroscopy. A good correlation (r = 0.96) was found between results from near-infrared spectroscopy and the Kjeldahl method. The imprecision of both methods was comparable. Once the rather laborious calibration has been performed, near-infrared spectroscopy is shown to be a very simple and rapid method for measuring nitrogen in faeces.

Determination of faecal fat by near-infrared spectroscopy.

The applicability of near-infrared spectroscopy to determine the amount of fat in faeces has been investigated. Near-infrared spectroscopy was favourably compared with the well known titrimetric method (Van de Kamer et al., J Biol Chem 1948; 177:347-55). A good correlation between near-infrared spectroscopy and the titrimetric method was found. The measurement of faecal fat by near-infrared spectroscopy is found to be more precise than the manual method. Moreover, near-infrared spectroscopy is shown to be a very simple and rapid method for measuring fat in faeces. However, it was shown that performing one's own calibration curve is necessary. Due to this necessity and the costs of the apparatus application of near-infrared spectroscopy is especially advantageous in laboratories with a substantial amount of samples to be analysed.

2',3'-dideoxyinosine (ddI): its chemical stability and cyclodextrin complexation in aqueous media.

The chemical stability of 2',3'-dideoxyinosine has been studied over a wide pH range (0-12). A stability-indicating HPLC method was used to separate the degradation product from the parent drug. The effects of temperature, ionic strength and buffer components on the degradation kinetics were investigated. Furthermore, the influence of some cyclodextrins (alpha-, beta-, HP-beta-, DM-beta- and gamma-cyclodextrin) on the drug stability have been studied.

Recognition and prevention of two cases of erroneous haemocytometry counts due to platelet and white blood cell aggregation. The use of acid citrate dextrose as an auxiliary anticoagulant.

Two cases of anticoagulant-induced platelet-white blood cell aggregation are described, which resulted in erroneous haemocytometry counts. Aggregation was avoided by the use of acid citrate dextrose (ACD) as an auxiliary anticoagulant, thus enabling quantification of platelets and white blood cells.

Recognition and prevention of pseudothrombocytopenia and concomitant pseudoleukocytosis.

Unrecognized anticoagulant-induced platelet (PLT) aggregation, leading to pseudothrombocytopenia and concomitant pseudoleukocytosis, can have serious clinical consequences. It can be readily recognized by inspecting conventional blood smears or the white blood cell histograms generated by modern blood cell counters. Blood specimens from twenty patients with known EDTA-induced platelet aggregation were consecutively drawn into three other anticoagulants (using Vacutainer tubes) and processed in a three-part differential Coulter Counter S Plus IV. PLT aggregation is shown to be generally induced by Li-heparin but much less frequently by citrate solutions. Prevention is almost invariably achieved by acid citrate dextrose (ACD). To that end approximately 2.5 mL of sterile ACD was aseptically injected beforehand through the stoppers of plain 5 mL Vacutainer tubes without breaking the vacuum. After blood drawing, correction for dilution was made by comparing hemoglobin values in EDTA and ACD.

Assay of urinary 5-hydroxyindole-3-acetic acid: two methodologies compared.

Udenfriend's (Sjoerdsma A, Weissbach H, Udenfriend S. J Am Med Assoc 1955; 159: 397) classical screening method for urinary 5-hydroxyindole-3-acetic acid (5-HIAA) is known to be subject to error due to food and drug interferences. Goldenberg (Goldenberg H. Clin Chem 1973; 19: 38-44) introduced a more specific and sensitive method, which has recently been modified by Zouheir Habbal (Zouheir Habbal M. Clin Chim Acta 1983; 130: 251-256). The latter method is shown to correlate fairly well with a high-pressure liquid chromatography (HPLC) method in the 0-100 mumol/24 h range (reference values up to 34 mumol/24 h). It is suggested that elevated values in Udenfriend's screening method can be reliably checked by applying either method.

Basic principles and problems of haemocytometry.

After some brief remarks on counting chambers, references to the ICSH-recommended haemoglobin-determination are given. The microhaematocrit of normal blood is advocated as a potential routine calibration method. Comments are given on discrepancies between centrifugal and flow haemocytometry haematocrits of abnormal and artificial bloods. Flow haemocytometry instruments are classified into analogue and digital instruments or into electrical and optical instruments. Their hydrodynamic properties are discussed. The principles and problems of electrical and optical cell counting and sizing are dealt with. The importance of the refractive index and of flow-induced cell shape changes for the MCV determinations is stressed. It is argued that MCV and haematocrit values are exaggerated at both low and high values and consequently MCHC is erroneously constant. Various prevailing red cell distribution width (RDW) and platelet distribution width (PDW) definitions bring about considerable confusion. The major features of the counting and sizing of white blood cells and platelets are described.

Comparison of the rheological behavior of hemocytometry controls and fresh patient EDTA-anticoagulated blood specimens.

For rheological reasons high viscosity specimens are more sensitive to deficiencies, if any, in the aspiration systems of whole blood flow cytometers and consequently are more likely to give erroneous values. For this reason the rheological behavior of hemocytometry controls was compared with that of patient blood specimens. It is shown that the behavior of controls and patient specimens of comparable hematocrit is similar at shear rates that are probably occurring during aspiration (exceeding approximately 40 s-1). The hematocrit is shown to be a major determinant of the viscosity of both controls and patient specimens. The results suggest that from the rheological point of view the fundamental IFCC premise is met, "that errors detected by means of control specimens exactly mirror errors occurring with patients' specimens", provided their hematocrits are taken into account.

Outdated blood and redundant buffy-coats as sources for the preparation of multiparameter controls for Coulter-type (resistive-particle) hemocytometry.

Outdated, buffy-coat depleted, CPDA-1 blood and redundant buffy-coats were used as sources for the laboratory preparation of controls for Coulter-type (resistive-particle) hemocytometry. Deteriorated white blood cells and platelets and potentially interfering microaggregates with volumes not exceeding 400 fl are shown to be virtually completely removed by centrifugation and filtration. Addition of fixed red blood cells as white blood cell substitutes and of isolated, fixed platelets enable the preparation of multiparameter controls of short-to-medium-term stability. The availability of these simple, inexpensive controls can contribute significantly to optimal internal quality control in hemocytometry.

Laboratory preparation and evaluation of a multiparameter hemocytometry control.

A protocol for the laboratory preparation of a multiparameter hemocytometry control is given. Human platelets, stabilized by a basically simplified and inexpensive fixation procedure, are added to our previously described white and red blood cell control. Evaluation of this multiparameter control shows good precision characteristics and acceptable mechanical stability for at least 7 weeks, as measured in the Coulter counter Model S Plus-II. The control can basically contribute to the realization of the essence of internal quality control: continuous self-auditing and continuous attempts at improvement of performance.

Sterile, medium scale age fractionation of human red blood cells.

After sterile removal of white blood cells and the majority of platelets from blood in a regular transfusion bag, the remaining red blood cells were separated under sterile conditions into a relatively young and a relatively old fraction. Making use of the different densities of old and young red blood cells, the separation occurs in a double transfusion bag by centrifugation, after the cells have been carefully layered on top of Percoll of "tailor-made" density. The successful results of this simple and inexpensive technique are assessed by comparing the creatine concentration and the acetylcholinesterase activity in both fractions. Thus, this medium scale fractionation technique yields hundreds of ml of sterile, young red blood cells.

Preparation and evaluation of a 7-parameter intralaboratory control blood of 4-month stability.

Sterile, fresh concentrates of human red blood cells suspended in a specially designed sterile preservation medium have a stability of all six red blood cell parameters of over four months as measured in the Coulter Model S-Plus II. Substitution under sterile conditions of the unstable white blood cells for (commercially available) fixed (human) red blood cells is a well-established method to obtain simulated white blood cell suspensions of long-term stability.

A stable human platelet-white blood cell control for the Coulter Model S-Plus II.

The preparation and evaluation of a human platelet-white blood cell control of at least 5 months' stability, as measured in the Coulter Counter Model S-Plus II, is described. In some preparations minor departures from log normal platelet (PLT) distributions were found, impeding the generation of values for mean PLT volume (MPV) and PLT distribution width (PDW). In many cases, however, excellent linearity for PLT and white blood cell (WBC) counts and low coefficients of variation for PLT and WBC counts and PDW were found. Some possible factors causing the large coefficient of variation found for MPV values are mentioned.

A simple, inexpensive quality control material for ortho ELT-8 platelet counts.

Dilutions of 1: 600 and 1: 3000 of 1 mum diameter polystyrene latex suspensions are simple, inexpensive materials for quality control of Ortho ELT-8 platelet counts. The coefficient of variation (CV) for a 12-week period at the 350 X 10(9)/l level was 2.2%, comparable with that of a commercial control. The CV at the 70 X 10(9)/l level was 3.5%.