Automated quantitation of hemoglobin-based blood substitutes in whole blood samples.

It is necessary to develop methods for accurate monitoring of cell-free hemoglobin in circulation. Routine monitoring of circulating cell-free hemoglobin will be useful for evaluating the efficacy of blood substitute administration andfor determining the clearance rates of the blood substitute from circulation. In addition, discriminating between cell-free hemoglobin and cell-associated hemoglobin will enable accurate determination of RBC indices, mean cell hemoglobin and mean corpuscular hemoglobin concentration, in individuals receiving hemoglobin-based blood substitutes. As colorimetric methods used by hematology analyzers to quantitate the hemoglobin value of a blood sample cannot distinguish between cell-associated and cell-free hemoglobin, it is currently not feasible to quantitate the levels of hemoglobin substitutes in circulation. The advent of a technology that measures volume and hemoglobin concentration of individual RBCs provides an alternative strategy for quantitating the cell-associated hemoglobin in a blood sample. We document that the combined use of cell-based and colorimetric hemoglobin measurements provides accurate discrimination between cell-associated and cell-free hemoglobin over a wide range of hemoglobin levels. This strategy should enable rapid and accurate monitoring of the levels of cell-free hemoglobin substitutes in the circulation of recipients of these blood substitutes.

Improved platelet counting using two-dimensional laser light scatter.

Clinical management of platelet disorders depends on accurate platelet counts. We evaluated a new analytic approach for platelet counting based on improved platelet discrimination. Current automated counting methods provide accurate platelet counts for most samples but often are unable to discriminate platelets accurately from nonplatelet particles such as microcytic RBCs, RBC fragments, and cellular debris that may falsely elevate platelet counts. The new approach measures 2 light-scatter angles of platelets and nonplatelet particles as they pass through a laser beam. The volume and refractive index of each platelet and particle are derived from the light-scatter measurements using the Mie scattering theory. Together, these 2 measurements provide improved platelet discrimination compared with 1-dimensional methods. With its improved discrimination, 2-dimensional platelet analysis provides more accurate platelet counts in samples containing interfering particles and may contribute to more effective clinical management of patients with platelet disorders.

Use of mean platelet component to measure platelet activation on the ADVIA 120 haematology system.

Platelet activation results in changes in a number of cell surface molecules including an increase in P-Selectin (CD62P) that may be rapidly and conveniently measured by immunofluorescent flow cytometry. The ADVIA 120 (Bayer) is a new system that facilitates more accurate measurement of platelet volume and in addition provides an approximate measure of the mean refractive index (RI) of the platelets reported as mean platelet component (MPC) concentration. We were interested to determine whether changes in MPC might reflect changes in platelet activation status. To investigate this, the platelet CD62P expression, determined by flow cytometry, and change in MPC, measured on the ADVIA 120 system, was first examined in vitro after stimulation of EDTA anticoagulated whole blood with submaximal concentrations of bovine thrombin in the presence or absence of the thromboxane synthase inhibitor, Ridogrel. Thrombin produced a dose-dependent increase in platelet CD62P expression and a decrease in MPC that could be inhibited by Ridogrel at physiological concentrations. In the second set of experiments, blood from 20 normal controls was collected into both EDTA and sodium citrate (SC) anticoagulants. Within 30 min of venesection and again at 3 h post-venesection after storage at room temperature, the platelet MPC and CD62P expression were determined. Platelets in all samples with both anticoagulants showed very low levels of CD62P expression when first analysed. At 3 h there was a small increase in CD62P expression on platelets in whole blood anticoagulated with SC, but a significant (P < 0.001) increase was observed on platelets anti-coagulated with EDTA. A negative correlation was found between the change in MPC of the platelets and the increase in the mean fluorescence intensity (MFI) (r = -0.69, P < 0.001, n = 20) and the percentage (r = -0.72, P < 0.001, n = 20) of CD62P positive platelets at 3 h in blood anticoagulated with EDTA. We conclude that a reduction in MPC as measured by the ADVIA 120 may be used to detect anticoagulant induced, as well as thrombin stimulated, in vitro platelet activation in blood anticoagulated with EDTA. Further, we conclude that platelet activation is negligible for up to 3 h in sodium citrate anticoagulated whole blood.

Reticulocyte hemoglobin content predicts functional iron deficiency in hemodialysis patients receiving rHuEPO.

Early detection of iron sufficiency at the level of the erythropoietic cell is necessary to optimize management of uremic anemia with recombinant human erythropoietin (rHuEPO). "Absolute" and "functional" iron deficiency are the most important factors causing resistance to administered rHuEPO. Transferrin saturation and serum ferritin measurements have been noted to be insensitive and inaccurate measures to detect functional iron deficiency. Recently, the reticulocyte hemoglobin content (CHr) has been shown to be a sensitive and specific indicator of functional iron deficiency in nondialysis patients treated with rHuEPO. The purpose of this study is to compare CHr with currently used indices of iron sufficiency in rHuEPO-treated hemodialysis (HD) patients. In study 1, 364 stable HD patients were studied at two outpatient dialysis centers. CHr was normally distributed, with a mean value of 28.3 pg, and was consistent over two consecutive monthly samples in each center. CHr was weakly but consistently correlated with transferrin saturation and serum ferritin. CHr and reticulocyte number were inversely correlated with red blood cell (RBC) number, suggesting that the erythropoietic stimulus of routinely administered rHuEPO may have resulted in functional iron deficiency. Month-to-month changes in CHr correlated weakly with changes in serum iron and percent transferrin saturation, but not at all with changes in serum ferritin. When we analyzed those patients with baseline CHr less than 26 pg, a level strongly suggestive of functional iron deficiency, these correlations strengthened, and in addition, month-to-month changes in CHr correlated strongly and directly with concomitant changes in RBC count, hemoglobin, and hematocrit, suggesting that rising CHr was indicative of an erythropoietic response. In study 2, 79 patients received a single-dose infusion of 500 mg iron dextran. After intravenous iron, CHr rose within 48 hours, peaked at 96 hours, and then fell toward baseline. Patients who were iron deficient by standard measures (serum ferritin < 100 ng/mL or transferrin saturation less than 20%) had a greater and a sustained CHr response to intravenous iron dextran. A CHr less than 28 pg at baseline predicted functional iron deficiency, defined as a corrected reticulocyte increase of greater than 1% to iron dextran, more accurately than transferrin saturation, ferritin, or their combination. Eighty-two percent of individuals who were iron deficient at baseline responded to intravenous iron with an increase in CHr of greater than 2 pg. Sixty percent of patients who were iron sufficient by usual iron indices also responded to intravenous iron with a CHr rise of greater than 2 pg, suggesting that they were, in fact, functionally iron deficient despite "normal" conventional iron parameters. We conclude that CHr may be a more sensitive marker of functional iron deficiency in rHuEPO-treated hemodialysis patients than percent transferrin saturation and ferritin, particularly in those with "normal" conventional iron parameters.

Reticulocyte hemoglobin: an integrated parameter for evaluation of erythropoietic activity.

Traditional reticulocyte counts provide only a partial estimate of the erythropoietic bone marrow activity and do not account for qualitative variations of reticulocyte cellular indexes and hemoglobin content in particular. We have studied a new integrated parameter, reticulocyte hemoglobin (retHb), that quantifies in grams per liter the hemoglobin contained in the circulating reticulocyte compartment and is obtained by multiplying the absolute reticulocyte count and the reticulocyte cell hemoglobin content. In 50 normal control subjects, retHb values were 1.76 +/- 0.59 g/L. The retHb values were lower in patients double heterozygous for HbS and HbC (SC disease) (3.33 +/- 1.52 g/L, n = 13) compared with homozygous HbS disease (SS) with concomitant alpha-thalassemia (5.27 +/- 1.51 g/L and 5.48 +/- 1.06 g/L for 12 patients with 3 alpha-genes and 3 patients with 2 alpha-genes, respectively) and to SS disease with no alpha-thalassemia (6.47 +/- 3.05, n = 20). The hemoglobin contained in the red blood cell pool (rbcHb) also can be calculated by subtracting retHb from the total hemoglobin. The ratio between the two pools (rbcHb/retHb, normal value 76.6 +/- 21.9, n = 50) provides a rough estimate of red blood cell survival. It was 9.8 +/- 4.1 in SS disease, 16.2 +/- 10.1 and 14.7 +/- 5.0 in SS disease with 3 and 2 normal alpha-genes, respectively, and 36.6 +/- 17.8 in SC disease with no alpha-thalassemia. We also studied retHb in patients receiving hydroxyurea therapy for SS disease, intravenous or oral iron for iron deficiency, or recombinant human erythropoietin (r-HuEPO) therapy. All these conditions are characterized by changes in reticulocyte counts and marked variations in reticulocyte cellular hemoglobin contents, which can be integrated into the retHb parameter. Measurement of retHb and the rbcHb/retHb ratio may provide an estimate of the reduction in red blood cell survival and the severity of hemolysis in various anemias and allow more precise monitoring of the response to hydroxyurea, iron, r-HuEPO, or other therapies.