Erythropoietic activities in acute leukemia and in malignant lymphoma with or without bone marrow involvement

Erythropoietic activities and immature reticulocyte production were investigated in a total of 157 patients (81 men, 76 women, median age = 42 yr, range = 23 to 65 yr) with acute lymphoid leukemia (ALL, n = 31), acute myeloid leukemia (AML, n = 39), or non-Hodgkin's lymphoma (NHL, n = 87), based on assays of the hemogram, red cell indices, reticulocyte subpopulations, and intramedullary erythroid precursors. There were no significant differences in red blood cell (RBC) counts, blood hemoglobin levels, or erythroid precursors between ALL and AML patients. Reticulocytes in AML patients averaged 1.7 +/- 0.8%, which was higher than in patients with ALL (0.8 +/- 0.3%, p <0.01); the proportion of high-fluorescence reticulocytes (HFR) averaged 4-fold higher in AML versus ALL (p <0.01) and the reticulocyte maturity index (RMI) was higher in AML (20.8 +/- 8.3%) versus ALL (12.4 +/- 6.5%, p <0.01). The RMI was higher in NHL patients with bone marrow (BM) involvement (15.6 +/- 9.4%), compared to those without BM involvement (4.3 +/- 2.1%, p <0.01). The proportion of HFR averaged 11-fold higher in NHL with BM involvement versus NHL without BM involvement. In summary, erythropoietic activity is significantly more active in patients with AML compared to ALL and in patients with NHL with BM involvement, compared to NHL without BM involvement.     

Associations between serum transferrin receptor concentrations and erythropoietic activities according to body iron status

This study investigated the associations between serum transferrin receptor (sTfR) concentrations and erythropoietic activities during 3 stages of iron deficiency in humans. Serum iron markers, fluorescent intensity of reticulocytes, and sTfR concentrations were measured in 227 prepubescent children, age 9 to 12 yr. Reticulocyte subpopulations were analyzed by flow cytometry and sTfR concentrations were measured by enzyme immunoassay. Mean values of middle-fluorescence reticulocytes (MFR), reticulocyte maturity index (RMI), and sTfR concentrations were significantly higher in iron-deficiency anemia subjects than in healthy controls. Reticulocyte subpopulations increased gradually, as body iron status diminished; the mean values of MFR and RMI in subjects with serum ferritin concentrations < 4.0 microg/L were 3-fold higher than those in healthy controls (p < 0.01). Correlation coefficients of MFR and RMI vs log ferritin values (r = 0.43 and r = 0.42) were higher than those of MFR and RMI vs sTfR concentrations (r = 0.24 and r = 0.27) in iron-deficiency anemia subjects. In summary, iron deficiency leads to increased production of immature reticulocytes. Erythropoietic activity is more closely associated with log ferritin values than with sTfR concentrations in iron-deficiency anemia.     

Reticulocyte as indication of the erythroid hematopoiesis: reticulocyte fractions in peripheral blood and bone marrow

Reticulocyte fractions of peripheral blood and bone marrow were measured for hematological disease in 235 patients using automated fluorescent reticulocyte analysis of Sysmex R-3000. The comparison with R-3000 and the manual method of bone marrow measurement showed an excellent agreement with a correlation coefficient of r = 0.933. In the ratio of reticulocyte fractions of marrow blood and peripheral blood, the marrow blood rate of reticulocytes, HFR, MFR, and LFR was 3.3, 6.2, 1.6, and 0.9 times higher than the peripheral blood rate, respectively. Cases in which the marrow reticulocyte rate was over ten times higher than the peripheral blood rate were observed in MDS and megaloblastic anemia at ratio of 55% and 100%, respectively. These findings suggest ineffective hematopoiesis in bone marrow. Immature reticulocyte fraction(10% or more) showed an excellent agreement with granulocyte(500/microliter or more) as an indication of the engraftment of allotransplantation of bone marrow, in which the analysis of reticulocyte fractions showed a useful indication of engraftment. In cases of death after bone marrow transplantation unlike in survivors, reticulocyte fractions decreased after engraftment.     

The correlation between hematopoietic status of the bone marrow and peripheral or marrow reticulocyte classification by using the automated reticulocyte analyzer Sysmex R-3000]

In this study peripheral and marrow reticulocytes were counted by using the automated reticulocyte analyzer Sysmex R-3000 with the quick and accurate function of reticulocyte classification dependent on reticulocyte maturation. The correlation of reticulocyte count of the R-3000 to that of visual reticulocyte counting method was r = 0.926, y = 0.88x + 0.305 and r = 0.933, y = 1.32x + 0.328 for peripheral blood and bone marrow, respectively. The hematopoietic status of the bone marrow was reflected in peripheral reticulocytes better than in leukocytes or platelets. Moreover, at recovery stage from bone marrow suppression after chemotherapy, the ratio of immature type (HFR fraction) in peripheral reticulocytes increased to precede in several days increasing total reticulocytes. The relative reticulocyte counts in bone marrow was about 3 times higher than peripheral blood to be drawn at the same time higher and the patients suffering from megaloblastic anemia, MDS or DIC tended to have a much higher reticulocyte count in bone marrow than in peripheral blood.     

Evaluation of the Sysmex R-1000. An automated reticulocyte analyzer

The new fully automated reticulocyte analyzer, Sysmex R-1000 (TOA Medical Electronics, Kobe, Japan), was evaluated for its routine use in the Hematological Laboratory at the University Hospital Basel, Switzerland. The operating characteristics, such as within-run precision, linearity, and carryover, fulfilled the manufacturer's specifications and are excellent. Correlation with the standard method, manual reticulocyte counting, is linear for normal and high values. For low reticulocyte counts the regression points show a deviation from their linearity. An absolute zero value is not obtained by the R-1000. The R-1000 measures total RNA content of each cell and expresses the value as low fluorescence ratio (LFR), medium fluorescence ratio (MFR), and high fluorescence ratio (HFR). The analysis of this ratio resolves the problem of zero reticulocytes: A fraction of less than 0.002 (0.2%) with an LFR of 100% represents aplasia; a shift of the intensity of fluorescence to HFR heralds regeneration. Results of samples stored at room temperature remain stable and within the range of the within-run precision for up to 12 hours, when stored at 5 degrees C for more than 48 hours. The authors conclude that the R-1000 is easy to operate, fulfills the criteria for accuracy and precision, and is highly suitable for daily routine use in a large central hematologic laboratory.     

血细胞多参数联合分析在大细胞性贫血疾病中的诊断价值

目的 探讨血细胞多参数联合分析在大细胞性贫血疾病中的临床诊断及鉴别诊断价值.方法 用Sysmex XE-5000型全自动血细胞分析仪对急性白血病(AL)组42例、溶血性贫血(HA)组30例、骨髓增生异常综合征(MDS)组27例、巨幼细胞性贫血(MA)组24例、再生障碍性贫血(AA)组16例,多发性骨髓瘤(MM)组15例及健康组50例的抗凝静脉血标本进行红细胞参数(HGB、MCV、MCH、MCHC、RDW-CV)、网织红细胞参数(Het％、LFR、MFR、HFR、Ret-He、IRF)及血小板相关参数(PLT、MPV、PDW、IPF)检测,并作统计分析大细胞性贫血患者各参数的分布规律及临床鉴别诊断.结果 ①所有疾病贫血患者组的RDW-CV值均高于正常对照组,HA组RDW-CV与其他疾病组间差异有统计学意义(P＜0.05).②所有疾病贫血患者组的MFR、HFR、IRF明显高于正常对照组,LFR低于正常对照组,HA组的MFR、HFR、IRF显著高于其他疾病组,差异有统计学意义(P＜0.05).AL、HA、MA、MM组的Ret-He均升高,MDS组与其他疾病组比较,Ret-He差异有统计学意义(P ＜0.05);MDS组与健康对照组比较,Ret-He差异无统计学意义(P＞0.05).③MDS组患者PDW值均高于其余各疾病组,差异有统计学意义(P＜0.05),与正常对照组比较,差异无统计学意义(P＞0.05).结论 血细胞相关参数是诊断大细胞性贫血疾病的重要依据,对于不同大细胞性贫血疾病的诊断及鉴别诊断具有十分重要的价值,为临床医生提供有方向地选择特异性诊断检查.     

Leukaemia and anaemia in AKR/O mice I. Leukaemia characteristics, haematological variables and erythropoiesis stimulating factor(s)

AKR/O mice were used as a model for studying the pathogenesis of the anaemia accompanying leukaemia/lymphoma. The leukaemia incidence was 87%. Median age at diagnosis was 11.3 months. At diagnosis most of the mice had normal leukocyte counts. Clinically the mice divided into subgroups depending on the relative organ involvement: 1) thymoma group (n = 98), 2) spleen group (n = 144), 3) combined group (n = 27) and 4) mice with moderate organ changes (n = 216). Mice of group 1 were younger than the others, had a rapidly progressive disease, normal to elevated packed cell volume (PCV), and plasma erythropoietin (Epo) was normal or increased. Mice of group 2 were usually anaemic with high plasma Epo estimates and often elevated reticulocyte counts. Group 4 was the oldest group. Some of these mice were severely affected haematologically. Overall there was an inverse relation between PCV and plasma Epo estimate, indicating a normal Epo response to anaemia. In all groups increasing spleen size was associated with increased severity of anaemia and increased reticulocyte counts. The association between anaemia, elevated reticulocyte counts and spleen enlargement suggests haemolysis as a mechanism for anaemia, and also raises the question of compensatory spleen erythropoiesis.     

Characterization of the myelotoxicity of chloramphenicol succinate in the B6C3F1 mouse

Chloramphenicol (CAP) is haemotoxic in man, inducing two types of toxicity. First, a dose-related, reversible anaemia with reticulocytopenia, sometimes seen in conjunction with leucopenia and thrombocytopenia; this form of toxicity develops during drug treatment. The second haemotoxicity is aplastic anaemia (AA) which is evident in the blood as severe pancytopenia. AA development is not dose-related and occurs weeks or months after treatment. We wish, in the longer term, to investigate CAP-induced AA in the busulphan-pretreated mouse. However, as a prelude to that study, we wanted to characterize in detail the reversible haemotoxicity of CAP succinate (CAPS), administered at high dose levels in the mouse, and follow the recovery of the bone marrow in the post-dosing period. Female B6C3F1 mice were gavaged with CAPS at 0, 2500 and 3500 mg/kg, daily, for 5 days and sampled (n = 5) at 1, 7, 14 and 21 days post-dosing. Blood, bone marrow and spleen samples were analysed and clonogenic assays carried out. At day 1 post-dosing, at both CAPS dose levels, decreases were seen in erythrocytes and erythrocyte precursors; marrow erythroid cells were reduced. Reductions were also evident in splenic nucleated cell counts, blood high fluorescence ratio (HFR) reticulocyte counts and total reticulocyte counts; burst-forming units-erythroid and colony-forming units-erythroid showed decreases. At day 7 post-dosing (2500 mg/kg CAPS), there was regeneration of erythrocyte production, with marked splenic erythropoietic activity, and raised blood HFR reticulocytes. At day 7, at 3500 mg/kg CAPS, erythrocyte and reticulocyte parameters remained depressed. At 14 days post-dosing (2500 mg/kg CAPS), many erythrocyte parameters had returned to normal; at 3500 mg/kg CAPS, there was erythroid regeneration. By 21 days post-dosing, at both CAPS dose levels, most erythrocytic parameters were equivalent to control values. For leucocyte parameters, there was some depression at day 1 post-dosing (at both CAPS dose levels) and signs of recovery at day 7. At days 14 and 21 post-dosing, most leucocyte parameters were close to control values. Marrow smears at day 1 post-dosing (at both CAPS dose levels) showed vacuolation of early normoblasts, of myeloid and of monocytic precursors. We conclude that the administration of CAPS at 2500 and 3500 mg/kg for 5 days induced significant myelotoxicity in female B6C3F1 mice, with cessation of erythropoiesis at day 1 post-dosing; recovery was seen over the following 7/14 days. The blood HFR reticulocyte count was a precise indicator of CAPS-induced depressive effects and subsequent recovery. It is concluded that the administration of five daily doses of CAPS at 2500 and 3500 mg/kg to the female B6C3F1 mouse induces an anaemia with reticulocytopenia, in conjunction with leucopenia, in the immediate post-dosing period; no evidence was seen at 21 days post-dosing of peripheral blood pancytopenia or a hypocellular/acellular bone marrow, which are both characteristic features of AA in man.     

Effect of iron status on reticulocyte mean channel fluorescence.

Flow cytometric reticulocyte enumeration measures the fluorescence intensity of the reticulocyte population, the reticulocyte mean channel fluorescence. Reticulocyte mean channel fluorescence, used as an indicator of reticulocyte maturation, is directly proportional to the amount of intracellular RNA. Other factors, such as iron stores, may affect reticulocyte mean channel fluorescence. Iron status in normal controls, patients with anemia of chronic disease, and pregnant women was evaluated by hemoglobin, hematocrit, red blood cell indices, iron, total iron-binding capacity, and ferritin. Reticulocyte mean channel fluorescence was significantly elevated (P less than 0.0001) to 85.6 +/- 4.6 (mean +/- 1 standard deviation) in iron-deficient anemic patients and to 81.1 +/- 8.4 in iron-depleted patients compared to healthy individuals (69.7 +/- 2.6). The reticulocyte mean channel fluorescence in anemia of chronic disease was 71.3 +/- 5.8 and was not significantly different from that of normal controls. Reticulocyte mean channel fluorescence showed significant correlations with total iron-binding capacity (P less than 0.0001, r = 0.62) and ferritin (P less than 0.0001, r = 0.40). A possible explanation for these findings, describing differences in cytoplasmic levels of transferrin receptor mRNA, is discussed.     

Automated reticulocyte counting and immature reticulocyte fraction measurement. Comparison of ABX PENTRA 120 Retic, Sysmex R-2000, flow cytometry, and manual counts.

We evaluated reticulocyte counting and measurement of immature reticulocyte fraction (IRF) with the ABX PENTRA 120 Retic blood analyzer on 300 blood samples. Reticulocyte counts were compared with those obtained by visual counting of 2,000 RBCs, by the TOA (Kobe, Japan) Sysmex R-2000 and a flow cytometry method. The parameters analyzed were the percentages of reticulocytes on all analyzers and the IRF with different modalities. The Retic Count kit (Becton Dickinson, San Jose, CA) was used with the Coulter (Hialech, FL) XL, and a mean channel of fluorescence (MCF) was calculated to fit the reticulocyte maturation. Reticulocyte counting with the ABX (Montpellier, France) PENTRA 120 Retic showed excellent precision and linearity with no significant carryover. Reticulocyte counts were stable after blood storage for 72 hours at 4 degrees C but not at room temperature (RT). IRF parameters values were stable for only 8 hours at 4 degrees C and 6 hours at RT. Comparisons of the methods showed good intraclass correlation (RI) for reticulocyte percentages between ABX PENTRA 120 Retic and Sysmex R-2000, ABX PENTRA 120 Retic and flow cytometry, Sysmex R-2000 and flow cytometry, and ABX PENTRA 120 Retic and manual counting. IRF values were correlated between fluorescence rates and RNA content, but in each case, low RI values were found, showing that Sysmex and ABX IRF values were not concordant. We obtained a significant correlation between mean fluorescence index and the MCF measured by flow cytometry, but the 2 methods were not concordant using the RI. The ABX PENTRA 120 Retic is a good instrument for analyzing reticulocyte count and percentage and allows a good analysis of IRF with several modalities.     

Reticulocyte measurements in rat, dog and mouse whole blood samples using the Sysmex XT-2000iV

Immature reticulocyte measurements in peripheral blood are being used as an indicator of erythropoiesis. Some haematology analysers can now provide an indication of reticulocyte maturity by dividing the reticulocytes into three counting regions based on fluorescent intensity or absorbance. In this study, reticulocyte measurements were evaluated for three laboratory species—rat, dog and mouse—and we found the Sysmex XT-2000iV to give satisfactory analytical performance in terms of precision, carry-over between samples, linearity and sample stability. Using the XT-2000iV, results were obtained for reticulocyte absolute counts and the three regional subpopulations of reticulocytes, and these were compared with results obtained with the Siemens Advia 120?. The study highlighted differences between the analysers which may reflect the methodology of the Advia 120? absorbance measurements and the fluorescence method used by the XT-2000iV. The different cell gating approaches and the lack of suitable calibration and control materials also contributes to these findings. It is important to establish baseline data before exploiting these measurements when investigating perturbations of erythropoiesis due to xenobiotics. We also investigated the correlations between the mean reticulocyte haemoglobin content (CHr), available on the Advia 120? and the two parameters (Ret-Y and RBC-Y) available on XT-2000iV, as both of these measurements can be used to investigate iron deficiency anaemias.     

Characterisation of busulphan-induced myelotoxicity in B6C3F1 mice using flow cytometry

Three experiments were carried out to investigate the myelotoxicity of busulphan in female B6C3F₁ mice using the Technicon H^*1 and the Sysmex R-1000 flow cytometers, instruments which produce a full blood count and a differential leucocyte count, and an automated reticulocyte count, respectively. In Experiment 1, a single dose of busulphan was administered at levels from 0 to 60 mg/kg and blood parameters measured at day 14. In Experiment 2, four doses of busulphan, from 0 to 40 mg/kg, were given at fortnightly intervals, and blood samples taken at days 14 and 42. In the third experiment, a single dose of busulphan was given at 0, 35 or 45 mg/kg and sequential blood, marrow and spleen samples examined up to day 10. In the first experiment there was a dose-related depression in the numbers of all leucocyte types. Values for Hb, RBC and HCT were not affected, whereas MCV and percentage macrocytic erythrocytes were increased, and MCHC was decreased, at high dose levels. Platelet numbers showed marked dose-related decreases. There were dose-related decreases in the numbers of all leucocyte types in Experiment 2 at days 14 and 42. Large unstained cell (LUC) numbers were reduced, and the mean neutrophil peroxidase index (MPXI) was increased, at high busulphan levels. Hb, RBC and HCT were reduced, whereas MCV, MCH and percentage macrocytic and percentage hypochromic erythrocytes were increased, in a dose-related fashion. Reticulocyte numbers showed a dose-related upward trend, but platelet counts illustrated a dose-related decrease, at days 14 and 42. In Experiment 3, busulphan caused a depression with a ‘U’-shaped curve, in the numbers of monocytes, eosinophils, lymphocytes and neutrophils. Decreased values and ‘U’-shaped curves were also seen for Hb, RBC, HCT and reticulocyte counts. Reticulocyte fluorescence ratio analysis showed that the high fluorescence ratio (HFR) was affected first and most profoundly. Calculation of the reticulocyte maturation index also demonstrated a dose-related effect on the earliest reticulocytes, and a ‘rebound’ effect. Total nucleated cell counts of the spleen and femur showed decreasing cell numbers and ‘U’-shaped responses with 45 mg/kg busulphan. This series of three experiments has established the use of a 6 week dosing regimen, with busulphan administered at fortnightly intervals, to induce myelotoxicity in a range of haematological parameters in female B6C3F₁ mice. We consider the use of the newly-developed flow cytometers and associated software, and the measurement of ‘non-standard parameters’ such as LUC, HFR and MPXI, to be particularly effective in the charcterisation of these busulphan-induced haematological changes.     

Reticulocyte maturation parameters are reliable early predictors of hematopoietic engraftment after allogeneic stem cell transplantation.

Early detection of donor-derived hematopoietic restoration after allogeneic stem cell transplantation (allo-SCT) is a crucial issue in the management of heavily immunocompromised patients. The aim of this prospective study was to validate our previously defined cutoff values for reticulocyte maturation parameters as early predictors of hematopoietic engraftment. Importantly, the effect of clinical variables in reticulocyte engraftment was also sought. For this purpose, we prospectively studied 136 consecutive patients undergoing allo-SCT from related (n = 89) or unrelated (n = 47) donors. High fluorescence reticulocytes (RETH), immature reticulocyte fraction (IRF), mean fluorescence index (MFI), and mean reticulocyte volume (MRV) were automatically measured in peripheral blood samples drawn on a daily basis. We previously defined reticulocyte engraftment when MFI > or =10, RETH > or =3%, IRF > or =10%, and MRV > or =110 fL. Median neutrophil engraftment was 18 days (range, 10-35 days); for reticulocyte parameters, the values were 14 days for IRF (range, 7-45 days), 14 days for MFI (range, 7-43 days), 15 days for RETH (range, 7-43 days), and 21 days for MRV (range, 9-74 days). These differences reached statistical significance for MFI and IRF when compared with standard neutrophil recovery, even when analyzing siblings or unrelated donors separately. In univariate analysis, donor-recipient ABO disparity adversely influenced erythroid engraftment (P = .04 for IRF, P = .03 for MFI), but the infusion of >2.9 x 10(6)/kg of CD34+ cells was associated with a shorter time to reach erythroid engraftment (P = .02 for IRF and MFI). In Cox regression analysis, > or =100/microL neutrophils and IRF > or =10% were predictive parameters for standard neutrophil engraftment. Based on these findings, we suggest that serial measurement of IRF or MFI should be routinely used to trace hematopoietic restoration after allo-SCT because these preceded standard neutrophil recovery by a median of 4 days and are therefore very useful to make clinical decisions.     

Flow cytometric reticulocyte quantification using thiazole orange provides clinically useful reticulocyte maturity index

Flow cytometric reticulocyte quantification with thiazole orange has been reported to be of potential utility in a clinical hematology laboratory. We have instituted this technique into routine clinical testing for 18 months and we describe this experience. Flow cytometric analysis provided not only reproducible, cost-effective reticulocyte quantification, but a quantitative reticulocyte maturity index proportional to the amount of RNA in the reticulocytes. The reticulocyte maturity index measurement represents an independent parameter of erythropoiesis, which provided clinically valuable information regarding bone marrow engraftment in patients following autologous bone marrow transplantation. The findings of this study demonstrate the clinical utility of thiazole orange reticulocyte analysis and indicate the diagnostic importance of the reticulocyte maturity index measurement in the evaluation of erythropoietic activity.     

An automated optoelectronic reticulocyte counter

Microscopic reticulocyte counting is time consuming and imprecise. A new reticulocyte counter has been developed, and the authors evaluated its utility for laboratory use. The counter, R-1000 of Sysmex-TOA Medical Electronics Company, Kobe, Japan, is based on the principles of flow cytometry. Reticulocytes are detected as fluorescent cells stained with a basic dye, auramine O, under argon-laser light. The automated count had high correlation to the manual count (r = 0.941). Linearity and reproducibility were both high. About 60 specimens were tested in one hour. Not only the reticulocyte percentage and count but also the maturity of reticulocytes was found from the intensity of the fluorescence, whether high, moderate, or slight. Normal reference values were 0.007 +/- 0.0055 (0.70 +/- 0.55%) for the reticulocytes, (4.63 +/- 1.09) X 10(9)/L for the reticulocyte count, 2.3 +/- 1.9% for highly fluorescent cells, 18.7 +/- 5.1% for moderately fluorescent cells, and 78.8 +/- 6.6% for cells with slight fluorescence. In patients with suppressed bone marrow function, such as is caused by chemotherapy, the reticulocyte fraction and count were low, and cells with slight fluorescence increased. In patients in whom bone marrow function was stimulated, such as with hemolytic anemia, the reticulocyte percentage, reticulocyte count, and highly fluorescent cells were high. Patients with chronic renal failure being treated by hemodialysis had a similar reticulocyte pattern to that in hemolytic anemia except that the reticulocyte count was decreased. Results for elderly patients were not different from those of healthy young controls. Some patients with a normal reticulocyte count and percentage had numerous highly fluorescent cells, perhaps because of hemolytic anemia not yet identified. Automated reticulocyte counting provides reliable data, so such measurement should be useful for analysis of the kinetics of red blood cells and for the study of the pathogenesis of anemia.     

Clinical utility of serum soluble transferrin receptor levels and comparison with bone marrow iron stores as an index for iron-deficient erythropoiesis in a heterogeneous group of patients

AIM: To evaluate the correlation between raised soluble transferrin receptor (sTfR) and stainable marrow iron, and to define the utility of sTfR in discriminating between the presence or absence of iron-deficient erythropoiesis in patients with anaemia of chronic disease. METHODS: Seventy-six consecutive adult patients without accelerated erythropoiesis who had undergone bone marrow (BM) aspiration/trephine for various clinical reasons during 2003-2006 were studied. All patients had serum iron studies (iron, transferrin and ferritin) and sTfR performed within 1 week of BM aspiration/trephine. These 76 patients were assigned to three groups based on the iron status of the BM and sTfR level: patients with normal sTfR and normal BM iron stores (n = 49), patients with an elevated sTfR and normal BM iron stores (n = 13) and patients reduced or absent BM iron stores (n = 14). Means (95% confidence interval) for mean corpuscular volume (MCV), mean corpuscular haemoglobin concentration (MCHC), red blood cell haemoglobin (RBC Hb) content and median (5th and 95th percentiles) for haemoglobin were then calculated. RESULTS: All patients with absent BM iron stores had an elevated sTfR level. Patients with normal BM iron stores and elevated sTfR levels had significantly lower Hb, MCV, MCHC and RBC Hb content than patients with normal BM iron stores and normal sTfR levels. CONCLUSION: sTfR is the most sensitive serum biochemical marker for the identification of iron-deficient erythropoiesis. Normal BM iron stores can coexist with elevated sTfR and decreased MCV and MCHC. sTfR levels correlate better than BM iron stores with decreased MCV and MCHC. Therefore, sTfR is a useful marker of iron-deficient erythropoiesis, due to both absent iron stores, and restricted iron supply due to anaemia of chronic disease. As a single investigation, however, sTfR does not discriminate between these two causes of iron-deficient erythropoiesis.     

Haematological parameters and altered erythrocyte metabolism in anaemic dogs

In previous studies, an increase in the activity of pyruvate kinase (PK) and glucose-6-phosphate dehydrogenase (G6PDH), an increase in the concentration of 2,3-diphosphoglycerate (2,3DPG), and alterations in osmotic fragility were found in dogs with haemolytic anaemia. These changes were mainly caused by the presence of immature red cells found in regenerative anaemias. In the present study, the same parameters were evaluated in dogs with different types of anaemia. The haematological patterns of 40 anaemic dogs were analysed to define the pathogenesis and the haematological features of each case. Non-regenerative anaemias could be attributed principally to chronic diseases and to the haemolysis that accompanies the early stages of canine babesiosis. Regenerative anaemias were mainly due to haemolysis, in some cases with an immune-mediated pathogenesis. PK activity was higher in regenerative than in non-regenerative anaemias, but G6PDH activity and 2,3DPG concentration increased in both types of anaemia. This suggests that PK activity is influenced by the presence of immature red cells, but the requirements for reducing compounds and oxygen are not dependent on the type of anaemia. Abnormalities in osmotic fragility were detected in haemolytic anaemias and in those non-regenerative anaemias in which reticulocyte percentage, but not reticulocyte production index (RPI), increased. The osmotic fragility could be used as an early indicator of erythrocyte regeneration.     

Characterisation of busulphan-induced myelotoxicity in B6C3F1 mice using flow cytometry

Three experiments were carried out to investigate the myelotoxicity of busulphan in female B6C3F₁ mice using the Technicon H^*1 and the Sysmex R-1000 flow cytometers, instruments which produce a full blood count and a differential leucocyte count, and an automated reticulocyte count, respectively. In Experiment 1, a single dose of busulphan was administered at levels from 0 to 60 mg/kg and blood parameters measured at day 14. In Experiment 2, four doses of busulphan, from 0 to 40 mg/kg, were given at fortnightly intervals, and blood samples taken at days 14 and 42. In the third experiment, a single dose of busulphan was given at 0, 35 or 45 mg/kg and sequential blood, marrow and spleen samples examined up to day 10.In the first experiment there was a dose-related depression in the numbers of all leucocyte types. Values for Hb, RBC and HCT were not affected, whereas MCV and percentage macrocytic erythrocytes were increased, and MCHC was decreased, at high dose levels. Platelet numbers showed marked dose-related decreases. There were dose-related decreases in the numbers of all leucocyte types in Experiment 2 at days 14 and 42. Large unstained cell (LUC) numbers were reduced, and the mean neutrophil peroxidase index (MPXI) was increased, at high busulphan levels. Hb, RBC and HCT were reduced, whereas MCV, MCH and percentage macrocytic and percentage hypochromic erythrocytes were increased, in a dose-related fashion. Reticulocyte numbers showed a dose-related upward trend, but platelet counts illustrated a dose-related decrease, at days 14 and 42. In Experiment 3, busulphan caused a depression with a U-shaped curve, in the numbers of monocytes, eosinophils, lymphocytes and neutrophils. Decreased values and U-shaped curves were also seen for Hb, RBC, HCT and reticulocyte counts. Reticulocyte fluorescence ratio analysis showed that the high fluorescence ratio (HFR) was affected first and most profoundly. Calculation of the reticulocyte maturation index also demonstrated a dose-related effect on the earliest reticulocytes, and a rebound effect. Total nucleated cell counts of the spleen and femur showed decreasing cell numbers and U-shaped responses with 45 mg/kg busulphan.This series of three experiments has established the use of a 6 week dosing regimen, with busulphan administered at fortnightly intervals, to induce myelotoxicity in a range of haematological parameters in female B6C3F₁ mice. We consider the use of the newly-developed flow cytometers and associated software, and the measurement of non-standard parameters such as LUC, HFR and MPXI, to be particularly effective in the charcterisation of these busulphan-induced haematological changes.Originally presented at ECCP 93.     

Reticulocyte analysis in systemic lupus erythematosus and chronic renal failure using flow cytometry

The number and maturation of circulating reticulocytes were measured in patients with systemic lupus erythematosus (SLE) and chronic renal failure (CRF) using an automated hematological analyzer (Technicon H*3 RTX) for their erythropoietic activities. Both SLE and CRF patients had increased reticulocyte numbers with a low degree of maturation. The SLE patients had no changes in mean reticulocyte corpuscular volume (MCVr) as compared to normal subjects (110.20 +/- 15.43 fl. in SLE and 110.39 +/- 5.09 fl. in normal), whereas CRF patients had significantly increased mean corpuscular reticulocyte volume (MCVr = 120.99 +/- 8.09 fl., p-value = 0.0019 as compared with normal). Three cases of SLE with nephrotic syndrome (NS) had high degree of MCVr (113.4, 125.0 and 133.1 fl., respectively). The renal involvement in SLE patients and CRF patients may associate with increased reticulocyte corpuscular volume.     

Automated measurement of reticulocyte count by flow cytometry. II: Analysis of the blood containing abnormal erythrocytes or giant platelets

We have examined the influence of erythrocytes containing inclusion bodies, nucleated red cells or giant platelets on the measurement of reticulocyte count by automated machine, R-1000. Correlation of the reticulocyte count between automated and conventional method was extremely good in the blood containing red cells with Jolly bodies, Pappenheimer bodies or basophilic stippling . However, correlation was poor when the sample contained the nucleated red cells. Reticulocyte count was decreased in the blood with significant amounts of nucleated red cells. Since nucleated red cells themselves are not counted as reticulocytes in the machine, this was considered to be due to increased young reticulocytes which frequently appeared with nucleated red cells. Both cold agglutinated red cells and giant platelets apparently influenced the reticulocyte count by the R-1000. These results suggest that red cells with Jolly bodies, Pappenheimer bodies or basophilic stippling do not influence the automatic counting of reticulocytes. Although nucleated red cells, cold agglutinated red cells and giant platelets affected the reticulocyte count, the machine shows abnormal flags in most of above cases (except highly agglutinated red cells), so that one can recount reticulocytes by conventional method. We conclude the machine can safely count the reticulocytes even in the blood containing abnormal red cells or platelets.