High serum folates and the simplification of red cell folate analysis.

Recent substantial increases in clinical blood folate concentrations are noted. Since red cell folates (RCF) are calculated from whole blood folates (WBF) by subtraction of the endogenous serum folate (SF) component, the reporting of clinical RCF results may be delayed because an ever increasing proportion (15%) of diagnostic SF levels are high (> 20 ng/ml) and need a repeat analysis. We evaluated 'plasma replacement' as a simple preanalytical procedure in which endogenous blood plasma is removed from red cells by washing and substituted with 'low-folate' plasma (serum) as an alternative conjugase (gamma-glutamyl carboxypeptidase) source for folate polyglutamate hydrolysis. Washed and conventional RCF assays compared well after both manual (n = 115, r = 0.98, y = 1x + 1.26) and automated washing of red cells (n = 170, r = 0.96, y = 0.96x - 0.73 ng/ml) and were not significantly different. The interassay reproducibility of folate results from washed blood samples was good (CV = < 6%). This novel 'plasma replacement' step halves the cost of a valid RCF assay by eliminating the need for endogenous SF analysis, and it expedites the reporting of clinical results.     

High serum folates and the simplification of red cell folate analysis

Recent substantial increases in clinical blood folate concentrations are noted. Since red cell folates (RCF) are calculated from whole blood folates (WBF) by subtraction of the endogenous serum folate (SF) component, the reporting of clinical RCF results may be delayed because an ever increasing proportion (15%) of diagnostic SF levels are high (> 20 ng/ml) and need a repeat analysis. We evaluated ‘plasma replacement’ as a simple preanalytical procedure in which endogenous blood plasma is removed from red cells by washing and substituted with ‘low‐folate’ plasma (serum) as an alternative conjugase (gamma‐glutamyl carboxypeptidase) source for folate polyglutamate hydrolysis. Washed and conventional RCF assays compared well after both manual (n=115, r=0.98, y=1x + 1.26) and automated washing of red cells (n=170, r=0.96, y=0.96x – 0.73 ng/ml) and were not significantly different. The interassay reproducibility of folate results from washed blood samples was good (CV=< 6%). This novel ‘plasma replacement’ step halves the cost of a valid RCF assay by eliminating the need for endogenous SF analysis, and it expedites the reporting of clinical results.     

Erythrokinetics in pernicious anemia

Quantitative measurements of the erythropoietic activity of the marrow, ofcirculating red cell production and destruction have been made in patients withpernicious anemia in relapse and during response to vitamin B₁₂ therapy.

Total erythropoietic marrow activity as reflected by turnover of heme components proceeds at a rate of approximately 3 times normal. The delivery ofviable red cells to the circulating blood, however, does not increase above normal. This would indicate that the greater portion of marrow activity is ineffective in terms of blood production. This marrow dysfunction coupled with anincreased rate of cell destruction of approximately 3 times normal is responsiblefor the anemia. Total erythropoiesis is somewhat less, and effective erythropoiesisconsiderably less, than that which may be expected of the normal marrow underthe sustained stimulus of anemia.

The reticulocyte count is shown to be an unreliable index of blood productionin untreated pernicious anemia due to loss of reticulum from cytoplasma ofmany red cells before their delivery into circulation.

During the response to vitamin B₁₂ the ineffective erythropoiesis is convertedto effective erythropoiesis, whereas total erythropoiesis remains unchanged.The rate of blood production during recovery is 3 to 4 times normal.

Submitted on January 23, 1956 Accepted on May 3, 1956     

Folate status in Italian blood donors: relation to gender and smoking

BACKGROUND AND OBJECTIVES: Folate deficiency in the general population is associated with a risk of cardiovascular disease and various cancers. The aim of this study was to evaluate folate status in Italian blood donors and its relationship with gender and smoking habit. DESIGN AND METHODS: A prospective study of 201 first visit donors (99 males and 102 females) was undertaken to evaluate folate status by measuring serum folate (SF) and red blood cell folate (RCF) levels and relating those with gender and smoking habit (100 smokers and 101 non-smokers). RESULTS: The rates of SF level less than 6.8 nmol/L and RCF less than 340 nmol/L were 9.9% and 25.3%, respectively in Italian blood donors. Mean RCF level was significantly lower (p<0.05) in females than in males and in smokers compared to non-smokers (p<0.001). The risk of reduced RCF levels in smokers was related to the number of cigarettes smoked per day, more than nine cigarettes increased the relative risk (RR) of low RCF level to 2.93 (95% C.I.: 1.34-6.41). INTERPRETATION AND CONCLUSIONS: This study suggests that folate deficiency, evaluated by RCF and SF levels, is widespread in Italian blood donors. Moreover, RCF values seem related to gender in non-smokers and modified by smoking habit, according to the cigarettes number smoked per day.     

Red Cell Changes in Top,Middle and Bottom Layer of Packed Red Cells in Vitamin B12 Deficiency

Surviving macroreticulocytes are released to peripheral blood during the first 4 d following a B₁₂ injection to vitamin B₁₂ deficient patients with grave anaemia. Marked increase in number of reticulocytes, together with corresponding reduction in reticulocyte size is noted in the top layer of the centrifuged packed red column from d 4 to 6 after medication. This ‘remodelling’ of the ‘stress-reticulocytes’ seems best explained by a division of the cells into 2 equal, surviving, normal-sized reticulocytes. The macroreticulocytes, which probably result from skipped division in the marrow, may thus complete their last cytoplasmatic division in the peripheral blood. The numerous schistocytes, which are found in the top layer of the centrifuged packed red cell column, disappear during the first few days after treatment, and it is assumed that these cells are fragments from the relatively few megalocytes which have reached the peripheral blood prior to medication. Macroreticulacytes seem to have a maximum deformability on d 5 after the B₁₂ injection, shortly after the cell division has started.     

Elevated serum homocysteine as a predictor for vitamin B12 or folate deficiency

Abstract: Tissue deficiency of vitamin B₁₂ and folate results in an increase in serum homocysteine (sHcy). We have measured sHcy in patients with reduced serum vitamin B₁₂ and/or red cell folate (RCF) to determine its usefulness as a discriminant for the diagnostic interpretation of reduced vitamin levels. Of 3846 patients who had serum vitamin B₁₂ and RCF assayed, 335 (9%) had reduced vitamin levels. Multivariate analysis showed a significant association between sHcy and serum creatinine (p = 0.0001), positive intrinsic factor (IF) antibody or neutrophil hypersegmentation (NHS) (p = 0.001), increased MCV (p = 0.014) and low RCF (p = 0.025) but no relationship with the level of serum vitamin B₁₂ or haemoglobin. After censoring the patients with renal impairment (n = 54), the distribution of the remaining 72 patients with elevated sHcy was 37/151 (25%) with low serum vitamin B₁₂ with or without low RCF and 35/130 (27%) with low RCF alone. sHcy correctly identified response to vitamin therapy in 33/35 (94%) patients who had adequate parameters to assess response. The positive predictive values of IF antibody/NHS, macrocytosis and/or low RCF for elevated sHcy were 100% and 34% respectively. Twenty-four percent of patients with a low serum vitamin B₁₂ and elevated sHcy had no abnormal haematologic parameters as determined by the routine laboratory staff. These data suggest that the usefulness of measuring sHcy in a routine diagnostic setting is limited and a careful review of the peripheral blood for macrocytosis and NHS plus determination of RCF may be a more cost-effective process than sHcy assay in most instances to determine the presence of tissue deficiency.     

Bone marrow sinus cell packing: a determinant of cell release.

Ultrastructural studies of erythropoietin effects on the bone marrow of control and hypertransfused (65 hct) mice revealed a decrease in adventitial cell cover of the sinus apertures in erythropoietin-treated animals. A more striking finding, however, was the marked inhibition of erythropoietin-induced reticulocytosis by hypertransfusion itself. Hypertransfusion of the erythropoietin-treated animals appeared to decrease the reticulocyte response by inhibiting reticulocyte response by marrow cords in addition to inhibiting erythroid proliferation. This inhibition of reticulocyte response was associated with clustering of reticulocytes around the marrow sinuses which were packed with red cells. Acute lowering of the hematocrit of erythropoietin-treated, hypertransfused animals to normal at the time of maximal reticulocyte response in control animals resulted in more than a twofold increase in reticulocytosis with 2 hr. It is suggested that (1) elevated levels of erythropoietin are associated with a diminution of the normal marrow-peripheral blood barrier, thereby contributing to the premature release of marrow elements and (2) the hematocrit is an important determinant of cell release from the marrow into the peripheral circulation.     

Red cell folate in elderly patients with myelodysplastic syndrome

Abstract: During a 7-month period a prospective study of 71 anaemic patients (29 males and 42 females) over the age of 50 was undertaken in order to identify patients with myelodysplastic syndrome (MDS). The mean values of mean corpuscular volume (MCV), serum ferritin, folate, vitamin B12 and red cell folate (RCF) of patients grouped according to the diagnosis were compared to those observed in age-matched blood donors. Forty-four of the 71 elderly patients showed macrocytic anaemia: 21 of them had gastric disease and the remaining 23 MDS. Two further patients with MDS showed microcytic anaemia. The 25 patients diagnosed with MDS were subclassified according to the FAB nomenclature: 9 had a refractory anaemia with excess of blasts and 16 refractory anaemia. The mean values of MCV, serum folate, ferritin, vitamin B₁₂ and RCF were statistically different between patients with macrocytic anaemia due to gastric disease and patients with MDS. Among patients with MDS, the RCF level pathologically high was inversely correlated to the haemoglobin level (r=-0.39; p<0.05). Thus the RCF and serum folate may represent useful parameters for the diagnosis of MDS in elderly anaemic patients.     

Simultaneous measurement of reticulocyte and red blood cell indices in healthy subjects and patients with microcytic and macrocytic anemia

Using the new Bayer H*3 hematology analyzer (Leverkusen, Germany), we have determined red blood cell and reticulocyte indices in 64 healthy subjects, in patients with microcytosis due to iron deficiency (58 patients) and heterozygous beta-thalassemia (40 patients), and in patients with macrocytosis (28 patients). We found in all cases that reticulocytes were larger than mature red cells by 24% to 35%, with a hemoglobin concentration 16% to 25% lower and a similar hemoglobin content. The correlation between red cell and reticulocyte indices was strikingly tight (r = .928 for volume, r = .929 for hemoglobin concentration, r = .972 for hemoglobin content) in all four groups, regardless of red blood cell size. The ratio of reticulocyte to red blood cell mean corpuscolar volume (MCV ratio) was constantly above 1. Inversion of the MCV ratio was observed only in four patients. It was always abrupt and transitory and was associated with erythropoietic changes leading to the production of red blood cells of a different volume (treatment of megaloblastic anemia, functional iron deficiency, bone marrow transplantation). In two cases of marrow transplantation, reticulocyte volume fell during the aplastic phase after conditioning chemotherapy and then rapidly increased up to values higher than before; this production of macroreticulocytes was the earliest sign of engraftment.     

Reticulocyte hemoglobin content

Under normal conditions, reticulocytes are the youngest erythrocytes released from the bone marrow into circulating blood. They mature for 1-3 days within the bone marrow and circulate for 1-2 days before becoming mature erythrocytes. Measurement of cellular hemoglobin concentration has long been reported by automated hematology analyzers as one of the red blood cell indices. The reticulocyte hemoglobin content (CHr or Ret-He) provides an indirect measure of the functional iron available for new red blood cell production over the previous 3-4 days. Measurement of reticulocyte hemoglobin content in peripheral blood samples is useful for diagnosis of iron deficiency in adults (Mast et al., Blood 2002;99:1489-1491) and children (Brugnara et al., JAMA 1999;281:2225-2230; Ullrich et al., JAMA 2005;294:924-930; Bakr and Sarette, Eur J Pediatr 2006;165:442-445). It provides an early measure of the response to iron therapy increasing within 2-4 days of the initiation of intravenous iron therapy (Brugnara et al., Blood 1994;83:3100-3101). Sequential measurements of reticulocyte hemoglobin content in patients with iron deficiency anemia provide a rapid means for assessing the erythropoietic response to iron replacement therapy (Brugnara et al., Blood 1994;83:3100-3101). It is also an early indicator or iron-restricted erythropoiesis in patients receiving erythropoietin therapy (Fishbane et al., Kidney Int 1997;52:217-222; Fishbane et al., Kidney Int 2001;60:2406-2411; Mittman et al., Am J Kidney Dis 1997;30:912-922; Tsuchiya et al., Clin Nephrol 2003;59:115-123; Chuang et al., Nephrol Dial Transplant 2003;18:370-377). Thus, reticulocyte hemoglobin content is a recent addition to an expanding list of biomarkers that can be used to differentiate iron deficiency from other causes of anemia.     

Erythrokinetics: quantitative measurements of red cell production and destruction in normal subjects and patients with anemia

Red cell turnover of 19 normal subjects and 25 anemic patients was measuredwith the following technique: erythroid-myeloid ratio of the marrow, reticulocytecounts, plasma iron turnover, red cell utilization of radioiron, and urobilinogendeterminations. Measurements of blood production and destruction were so expressed as to allow comparison between normal and anemic individuals of different size and different red cell mass. The usefulness and disadvantages of eachprocedure in the study of anemia are discussed.

From studies of various types of anemia, it has become apparent that erythropoiesis must be defined in terms of total quantity of red cells produced and interms of the portion of red cells produced in the marrow which are delivered tothe circulating blood (effective versus ineffective erythropoiesis). A quantitativedefect alone exists when a normal ratio is maintained between effective andtotal erythropoiesis. Here, there are changes of similar magnitude of all erythrokinetic indices, although reticulocyte and urobilinogen values are occasionallydisproportionately high. The normal marrow appears to be able to increase itseffective red cell production to three times normal in acute anemia and six timesnormal in chronic anemia. In many disease states this maximal quantitativeresponse is impaired.

Dyspoiesis of the marrow is characterized by a dissociation of erythrokineticindices. Values which reflect total erythropoiesis (i.e., plasma iron turnover,fecal urobilinogen and erythroid-myeloid ratio of the marrow) are considerablygreater than the reticulocyte level and red cell utilization of radioiron whichrepresent effective erythropoiesis. Such defects may result in the pattern of ahemolytic process or aregenerative anemia, depending on their severity.

Submitted on October 26, 1955 Accepted on December 7, 1955     

Megaloblastic anaemia in vegetarian Asians

Of 27 Asians with a megaloblastic bone marrow, 22 (81%) had nutritional deficiency of vitamin B12 (NMA), while five (19%) had true pernicious anaemia (PA). All the patients were Hindu vegetarians except for a single Muslim who had PA. Dietary intakes of calories, protein, iron, vitamin B12 and folate were below the recommended level in both groups. The PA group had lower levels of serum B12 and higher levels of serum folate than the NMA group. Despite low levels of red cell folate (RCF) in the NMA patients, the abnormality in deoxyuridine (dU) suppression was always corrected by vitamin B12. The dU suppressed value showed a significant inverse relationship to the RCF level. Nutritional deficiency of vitamin B12 is the most common cause of megaloblastic anaemia in Hindu vegetarians but the incidence of true PA is higher than previously thought and may approximate to that of the white population.     

The Maturation Rate of Reticulocytes

An in vitro culture technic for the study of reticulocyte maturation wasdescribed. The method gave reproducible results and proved to be of valuein the comparative study of reticulocyte maturation in blood disorders. By thismethod it was shown that variations in the reticulocyte maturation in vitroparalleled similar variations present in vivo.

The maturation of reticulocytes from patients with different types ofanemia was investigated. In some anemias the in vitro maturation of reticulocytes was prolonged, not only because younger reticulocytes were present inthe blood, but also because the rate at which the reticulum substance disappeared was delayed. This was particularly evident in the anemia of chronicuremia, in Cooley’s anemia and in pernicious anemia in relapse. In only occasional cases of hereditary spherocytosis and of autoimmune hemolyticanemia was the rate of reticulocyte maturation found to be moderatelydelayed. In patients with iron deficiency anemia or bleeding anemia it wasalways normal.

From the above findings the following conclusions were derived:

1. The reticulocyte number in the circulating blood is the resultant of threevariables: (a) the rate of output of new reticulocytes from the bone marrow;(b) the stage of maturation at which reticulocytes are delivered into theperipheral circulation; (c) the rate of disappearance of the reticulum substance.

2. The number of reticulocytes in the circulating blood cannot be indiscriminately used as a precise index of red cell production in erythrokinetics.

3. There is good reason to believe that a defect in the rate at which thereticulocytes mature in the circulating blood is an index of a similar defectin the process of erythroblastic differentiation in the bone marrow.

     

Mechanism of arsenic-induced inhibition of erythropoiesis in mice

Anemia accompanies arsenic intoxication in man. The present studies were undertaken to clarify further the effects of arsenic on erythropoiesis. A dose-related inhibition of red cell ⁵⁹Fe incorporation and reticulocyte response was observed in normal mice treated with a single injection of arsenic. Arsenite was approximately two times as inhibitory as arsenate. The effects of arsenic on erythropoietin-induced erythroid differentiation revealed a significant inhibitory effect on young, proliferating marrow nucleated erythroid precursor cells. More mature, nonproliferating nucleated erythroid cells were resistant to the toxic action of arsenic. A dose-related inhibitory effect of arsenic on DNA synthesis was observed in fetal liver nucleated erythroid cells incubated with ³H thymidine. Ineffective erythropoiesis as well as the megaloblastic morphology accompanying aberrant DNA synthesis – manifestations of arsenic toxicity in man – were not evident in the present studies.     

Effect of folic acid and vitamin C supplementation on folate status and homocysteine level: a randomised controlled trial in Italian smoker-blood donors

BACKGROUND: This trial sought to examine the effects of high dosage of folic acid and vitamin C supplementation on red blood cell folate (RCF), serum folate (SF) and homocysteine (Hcy) levels in subjects who smoke more than 15 cigarettes per day. METHODS: A prospective study of 100 Italian repeat blood donors was undertaken to measure RCF, SF and Hcy levels before and after 45 days of vitamin supplementation. All subjects were randomised into four groups: [A] folic acid (FA) 5 mg/day, [B] vitamin C 500 mg/day, [C] FA 5 mg/day plus vitamin C 500 mg/day [D] no supplementation. RESULTS: Before supplementation the median RCF, SF and Hcy levels were similar in the four groups; 32 (40%) subjects had an RCF level below 340 nmol/l, 15 (18.8%) had an SF level below 6.8 nmol/l and 21 (26.3%) had an Hcy level above 16 micromol/l. After 45 days the median RCF and SF levels were significantly (P<0.01) increased in all supplemented subjects. The median Hcy level was significantly (P=0.008) reduced in subjects supplemented with FA and significantly (P=0.01) increased in those supplemented with vitamin C alone. CONCLUSION: The supplementation with 5 FA mg/day is able to increase significantly both RCF and SF levels and reduce Hcy level in Italian smoker-blood donors.     

The effect of anemic anoxia on the cellular development of nucleated red cells

The action of an anoxic stimulus on red cell production was studied inrabbits bled 20 ml./Kg., kept anemic for 20 hours and then reinfused with thepreviously removed blood. This 20-hour period of anemic anoxia was followedby a characteristic reticulocyte response, a response which was modified bynitrogen mustard or colchicine administered immediately after the 20-hourperiod of anemia, but was not influenced by anoxia or hyperoxia in the postanemic period. When mitotic division was arrested by colchicine during the20-hour period of anemic anoxia, the onset of the reticulocyte response,though delayed by 1 to 2 days, was otherwise of characteristic magnitude.

These observations indicate that (1) the anoxic stimulus operates in thebone marrow by accelerating the differentiation of stem cells into pronormoblasts, and that thereafter (2) the maturation and multiplication of differentiated nucleated red cells proceed at fixed rates independent of the anoxicstimulus.

Submitted on June 18, 1958 Accepted on July 29, 1958     

Evaluation of iron stores in patients with alcoholic liver disease: role of red cell ferritin

Forty-two male patients with alcoholic liver disease were studied for iron status by indirect hematological assays, including red cell ferritin (RCF), and histochemical estimations. Serum iron and ferritin, total iron-binding capacity levels were unrelated to iron deposits, whereas RCF concentration was a good index of iron stores as detected by direct assessment on bone marrow and liver biopsy specimens. A relatively high proportion of alcoholics (19%) were iron-deficient. Alcoholic patients with cirrhosis exhibited higher RCF values than patients with alcoholic hepatitis. However, this increase was apparently unrelated to cirrhosis per se. In alcoholics we found that RCF was mainly related to levels of bone marrow iron. The increased RCF values observed in patients with hepatic siderosis was mediated by marrow iron stores. RCF can therefore be regarded as a useful test to distinguish patients with liver siderosis and normal values of bone marrow iron.     

On the diagnosis of erythrocyte enzyme defects in the presence of high reticulocyte counts

The separation of red blood cells into reticulocytes and young and old erythrocytes enables investigations of fractions with different contents of reticulocytes. Activities of hexokinase, glucose phosphate isomerase, phosphofructokinase, pyruvate kinase and glucose-6-phosphate dehydrogenase showed a linear relationship to reticulocyte counts. The dependence of these enzyme activities on the age of the red blood cells exhibited a strong decline from the reticulocyte to the young erythrocyte stage followed by only little further loss of activity, thus leading to a biphasic decay of enzyme activities. By linear regression analysis enzyme activities in erythrocytes (AE) and reticulocytes (AR) could be evaluated. The activity of a given enzyme in the reticulocyte exceeded that of the erythrocyte; the quotient AR/AE represents the decline of enzyme activity from the reticulocyte to the erythrocyte stage. This value AR/AE is 16.7 for pyruvate kinase and 9.4 for hexokinase and thus considerably higher than that for the other enzymes investigated (glucose phosphate isomerase: 2.9, phosphofructokinase: 4.3, glucose-6-phosphate dehydrogenase: 4.5). In patients suffering from erythrocyte enzymopathies, the AR/AE for pyruvate kinase was 16.2 and thus almost identical to the normal enzyme. Calibration curves where the enzyme activity is plotted versus the fraction of reticulocytes enable the determination of normal activity of a given erythrocyte enzyme depending on the content of reticulocytes in red blood cell suspensions. Thus an unambiguous diagnosis of enzyme defects irrespective of reticulocyte counts becomes possible.     

Reduced progression of atherosclerosis in apolipoprotein E-deficient mice with phenylhydrazine-induced anemia.

Epidemiological and experimental studies suggest that circulating erythrocytes play a role in the incidence of coronary heart disease. We investigated the influence of phenylhydrazine (PHZ)-induced anemia on the formation of atherosclerotic lesions in apo E-deficient mice on regular chow and on a high-fat, high-cholesterol diet during 10 weeks. The repeated doses of PHZ caused sustained anemia throughout the study, changes in the physical characteristics of erythrocytes and increased reticulocyte count. The lesions of the anemic animals were smaller than in the controls and this was even more evident in mice fed with the atherogenic diet. A positive correlation was found between circulating red blood cells at the end of the experiment and the area of aortic lesion. There was also a negative association between the lesion and the reticulocyte count. This reduced progression of atherosclerotic lesions is independent of nutritional status or the lipoprotein cholesterol distribution. The results suggest that mechanisms related to the number of circulating red blood cells may have a significant influence on the development of atherosclerosis.     

Adaptation of Marrow Sinus Wall to Fluctuation in the Rate of Cell Delivery: Studies in Rabbits after Blood-letting

The extent of marrow sinus wall coverage by perisinal adventitial cells was measured before and after removing 25% of the calculated blood volume in rabbits. A marked reduction in the area covered by adventitial cells was recorded coinciding with the early reticulocyte response to phlebotomy. In control animals, only scattered microfilaments were occasionally observed in the cytoplasmic process of adventitial cells. After phlebotomy, these filaments were more frequently observed and appeared as band-like structures, raising the possibility of an active role for perisinal adventitial cells in response to fluctuations in the magnitude of cell traffic. Perisinal adventitial cells may, therefore, translate the body's demand for cell delivery by regulating the rate of transmural cell migration from the marrow. Such an adaptive mechanism may depend on the incompleteness of the adventitial layer of the sinus wall and the absence of endothelial-adventitial cell junctions, permitting separation of the two layers of the wall, so that the reticulocyte can gain access to more endothelial surface and the pores.