Changes in Erythroblast Morphology as an Index of Response to Cyanocobalamin in Patients with Megaloblastic Anaemia

Fifty-three patients with megaloblastic anaemia treated with cyanocobalamin and folic acid have been studied. Repeat marrow examination was found to be of value in assessing response to treatment. The early improvement in marrow morphology in patients with pernicious anaemia was greater with 1000 μg than with 5 μg doses of cyanocobalamin. The effect of folate deficiency in delaying marrow response to cyanocobalamin in patients with pernicious anaemia is described and combined cyanocobalamin and folic acid treatment was found to be more effective than either alone. The response to large doses of cyanocobalamin in folate deficient patients was unrelated to the initial serum vitamin B₁₂ level.     

Marrow Cells from Patients with Untreated Pernicious Anaemia Cannot Use Tetrahydrofolate Normally

Folate analogues were added to human bone marrow cells to determine their effect on deoxyuridine utilization in the deoxyuridine suppression test. Forniyltetrahydrofolates fully corrected the impairment of dU utilization in pernicious anaemia marrows but tetrahydrofolate was relatively ineffective. All these analogues were effective in megaloblastic marrows from folate deficient patients.
Formyltetrahydrofolates also enhanced dU utilization by normal human marrows whereas methyltetrahydrofolate reduced its use.
In terms of the methylfolate trap hypothesis, the expectation that cobalamindeficient marrows would be able to use tetrahydrofolate normally was not realized.     

A Comparison of Tetrahydrofolate and 5-Formyltetrahydrofolate in Correcting the Impairment of Thymidine synthesis in Pernicious Anaemia

5-formyltetrahydrofolate and tetrahydrofolate were added to marrow cells from patients with untreated pernicious anaemia at 1, 5 and 50 nmol doses in the deoxyuridine suppression test. At all 3 dose levels formyltetrahydrofolate was significantly more effective in correcting the defect of thymidine synthesis in pernicious anaemia, than tetrahydrofolate. The data suggest that formylation of tetrahydrofolate is necessary for its normal utilization.     

Forms of Vitamin B12 in Blood and Bone Marrow in Patients with Pernicious Anaemia

The biologically-active forms of vitamin B₁₂ in blood and bone marrow and changes induced in these by injections of cyanocobalamin have been measured in patients with pernicious anaemia. Bone marrow methylcobalamin was low before therapy, and increased 24 h after therapy. The largest portion of bone marrow vitamin B₁₂ was 5′deoxyadenosylcobalamin, and this increased more than did methylcobalamin during the 24 h after injection of cyanocobalamin. A single injection of roo μ of cyanocobalamin induced about 10 times the increase of the intracellular coenzyme forms of vitamin B₁₂ in bone marrow than followed injection of 100 μg. Plasma methylcobalamin was extremely low before therapy, and increased only moderately 24 h after therapy; the majority of plasma vitamin B₁₂ remaining as cyanocobalamin. In contrast, only a minority of intracellular bone marrow vitamin B₁₂ was cyanocobalamin 24 h after injection of cyanocobalamin. The degree of anaemia did not correlate with bone marrow methylcobalamin, nor did bone marrow cobalamin correlate significantly with cobalamin content of washed blood erythrocytes. Correlation was observed between intra-erythrocyte vitamin B₁₂ content and the degree of anaemia; the correlation being inverse with haemoglobin concentration in the peripheral blood. Inverse correlation also was observed between MCV and erythrocyte folate content. These studies suggest that megaloblastic maturation appears at different concentrations of bone marrow vitamin B₁₂ in different patients, presumably because in vitamin B₁₂ deficiency, eventual limitation of normoblastic maturation may be determined by factors such as folate metabolism, vitamin B₁₂ binders, and the affinity of vitamin B₁₂ dependent enzymes. Although the clinical response to 1000 μg of cyanocobalamin does not differ from that to 100 μg, the concentration of vitamin B₁₂ coenzymes in bone marrow cells was proportional to the cyanocobalamin injected.     

Serum Ferritin in Megaloblastic Anaemia

Serum ferritin concentrations have been estimated in 30 patients with untreated megaloblastic anaemia, 27 with Addisonian pernicious anaemia. A significant difference was found between the mean serum ferritin level of the 27 pernicious anaemia patients (330 μg/l) and of 22 normal control subjects (164 μg/l) (P < 0.05 > 0.02). There was an inverse correlation between serum ferritin and Hb concentration in men with pernicious anaemia but not in women. Serum ferritin levels were lower in 10 of 13 patients studied after 24 h of vitamin B₁₂ therapy and in all 13 studied at 48 h after therapy. The fall continued during the haematological response to therapy. It seems likely that serum ferritin reflects reticuloendothelial iron and the high levels in untreated megaloblastic anaemia are due to the shift in iron from Hb to reticuloendothelial stores. The wide variation in serum ferritin at any given Hb level presumably reflects variation in iron stores of the individual patient.     

Folate-dependent Serine Synthesis in Lymphocytes from Controls and Patients with Megaloblastic Anaemia: the Effect of Therapy

The utilization of [¹⁴C]formate for serine synthesis by lymphocytes was impaired in all the patients with pernicious anaemia and in 70% of other patients with megaloblastic anaemia. In pernicious anaemia this was corrected by vitamin B₁₂ therapy in 48 h but not by folate therapy although patients given folate showed a satisfactory haematological response.     

Metabolism of 5-MethyItetrahydrofolate in Pernicious Anaemia

S ummary 5-Methyltetrahydropteroylglutamic acid was given intravenously to control subjects and to patients with untreated megaloblastic anaemia. The rate of clearance of this substance from the plasma and excretion into the urine after an intravenous dose of 5 μg./kg. of body weight was followed.
The rate of clearance was faster in patients with folate deficiency as compared to control subjects. In pernicious anaemia the clearance was either within the normal range or more rapid than normal if there was associated folate deficiency. No support was obtained for the hypothesis that in pernicious anaemia there is a failure to utilize 5-methylfolate.     

Severe megaloblastic bone marrow change associated with unsuspected mild vitamin B12 deficiency

Summary Two patients are reported who developed peripheral blood abnormalities and marked megaloblastic bone marrow change within eleven days of cardiac bypass surgery. The patients were shown to have unsuspected mild vitamin B₁₂ deficiency due to Addisonian pernicious anaemia. The megaloblastic changes were presumed to be precipitated by the increased demand for erythrocytes and platelets after surgery.     

The Effect of Deoxyuridine, Vitamin B12, Folate and Alcohol on the Uptake of Thymidine and on the Deoxynucleoside Triphosphate Concentrations in Normal and Megaloblastic Cells

Deoxyuridine suppression of labelled thymidine uptake tests were performed in the bone marrows of 58 patients with megaloblastic anaemia (haemoglobin less than 10.0 g/dl) and invariably gave values (range 10.3-58.8%) above the range in 16 control marrows (range 1.0-9.0%). Folinic acid corrected the test equally well in either folate or vitamin B12 deficiency, even at concentrations as low as 60 ng/ml. Folic acid also corrected the test equally well in either deficiency but was only effective at concentrations down to 5 microgram/ml. Vitamin B12 (100 microgram/ml) only corrected the test in vitamin B12 deficiency and 5-methyltetrahydrofolate only corrected the test in folate deficiency at the concentrations tested between 60 and 1.2 microgram/ml. Among 16 patients with subnormal serum levels of both vitamin B12 and folate, vitamin B12 partially corrected the test in eight, including all five with pernicious anaemia, but had no effect in the other eight. Despite the clear-cut results of the dU suppression test, measurement of the deoxythymidine triphosphate (dTTP) concentration in normal and megaloblastic phytohaemagglutinin stimulated lymphocyte cultures or short-term bone marrow cultures gave no clear-cut differences between normal and megaloblastic cells after addition of deoxyuridine nor did the addition of vitamin B12, folic acid or folinic acid either alone or with deoxyuridine produce consistent changes in the dTTP concentration in lymphocytes or bone marrow cells in megaloblastic anaemia. Alcohol caused a rise in deoxyadenosine triphosphate concentration in normal PHA-stimulated lymphocytes which was concentration dependent but caused no consistent change in any of the other three deoxynucleoside triphosphate (dNTP) concentrations. Diphenylhydantoin (10(-3)M, 10(-4)M) had no consistent effect on any of the four dNTP concentrations.     

Bone marrow status of anaemic pregnant women on supplemental iron and folic acid in a Nigerian community

The bone marrow status of 31 consecutive pregnant women who had been on supplemental oral iron and folic acid since early pregnancy at the University of Benin Teaching Hospital was assessed later in pregnancy to test the efficacy of oral iron and folic acid in preventing iron deficiency and/or megaloblastic anaemia in our community. Only those pregnant patients with haemoglobin genotype AA or AS took part in the study. Nobody was excluded except those with CC or SC. 96.77% (30 out of 31 patients) had iron deficiency with no stainable iron in the bone marrow. 35.4% (11 out of 31 patients) had megaloblastic changes in the bone marrow. 32.2% (10 out of 31 patients) had a combined iron deficiency and megaloblastic anaemia while only one out of 31 patients (3.23%) had megaloblastic anaemia without concurrent iron deficiency. 60.4% (20 out of 31 patients) had iron deficiency alone without concomitant megaloblastic changes in marrow. The bone marrow in all the patients were normal in other respects except with regards to iron-deficiency and/or megaloblastic status. The significance of this high incidence of iron-deficiency and/or megaloblastic anaemia in patients already on routine pre-natal drugs is discussed.     

Pernicious Anaemia in Indian Immigrants in the London Area

Two out of 15 cases of megaloblastic anaemia in Punjabi immigrants in the Southall area of Middlesex, studied in one year, were found to have all the features of Addisonian pernicious anaemia. This unexpected finding in Asiatics calls for a reappraisal of the previously accepted rarity of this disease in dark-skinned races and draws attention to the need for consideration of pernicious anaemia as a cause of megaloblastic anaemia in the Indian immigrant.     

Cyanocobalamin, Ascorbic Acid and Pteroylglutamates in Normal and Megaloblastic Bone Marrow

The B₁₂ activity as estimated by Lactobacillus leichmannii, the folic-acid-like activity by Streptococcus faecalis (F.A.A.) and the ascorbic acid concentration have been determined in the blood and buffy coat of bone marrowof normal subjects, 10 patients with pernicious anemia in relapse, a group ofpatients with non-Addisonian megaloblastic anemia and some patients withiron deficiency.

A correlation between the serum B₁₂ and the plasma ascorbic acid andtheir respective levels in bone marrow was observed. The marrow and serumB₁₂ levels in prenicious anemia were abnormally low, but they did not differfrom a group of 5 patients with hypochromic normoblastic anemia who hadboth low serum and marrow levels. The concentration of F.A.A. in the marrowof patients with pernicious anemia was reduced, but it was felt that this wasmore likely a manifestation of the megaloblastic anemia rather than a causativefactor.

One of six patients with megaloblastic anemia of pregnancy had no detectabledeficiency, while the other five had reduced B₁₂, folic acid and ascorbic acidconcentrations. The possible therapeutic implications are discussed.

There was a significant reduction in the bone marrow concentration ofascorbic acid in all patients with megaloblastic anemia.

Submitted on March 26, 1959 Accepted on October 9, 1959     

Leucocyte Folate in Vitamin B12 and Folate Deficiency and in Leukaemia

Leucocyte folate concentrations were measured in 24 normal subjects, in 32 patients with subnormal serum folate concentrations and normal serum B₁₂ concentrations associated with chronic gastro-intestinal disease, in seven patients with leukaemia, and in 10 patients with untreated pernicious anaemia.
In the normal subjects, the leucocyte folate levels ranged from 60 to 123 ng./ml. of packed leucocytes. Among the patients with subnormal serum folate concentrations, leucocyte folate concentrations were normal in seven or eight patients with entirely normoblastic haemopoiesis, but were subnormal in all eight patients whose marrows showed normoblastic erythropoiesis and giant metamyelocytes, in all eight patients with obvious megaloblastic changes, and in all eight patients with overt megaloblastic anaemia due to folate deficiency.
Leucocyte folate was raised in six of the seven patients with leukaemia including patients with acute and chronic myeloid leukaemia, myelomonocytic leukaemia and chronic lymphatic leukaemia.
Of the 10 patients with untreated pernicious anaemia, three had raised, three had normal, and four subnormal leucocyte folate. The changes in leucocyte folate produced by B₁₂ therapy in four of the pernicious anaemia patients are described.     

Vitamin B12 deficiency is the primary cause of megaloblastic anaemia in Zimbabwe

Summary. In a study of the pathogenesis and clinical features of megaloblastic anaemia in southern Africa, we evaluated 144 consecutive Zimbabwean patients with megaloblastic haemopoiesis. Vitamin B₁₂ deficiency was diagnosed in 86.1% of patients and was usually due to pernicious anaemia; isolated folate deficiency accounted for only 5/5% of cases. Anaemia was present in 95.8% of patients; the haemoglobin (Hb) was 6 g/dl in 63.9%. Neurological dysfunction was noted in 70.2% of vitamin B₁₂-deficient patients and was most striking in those with Hb values > 6 g/dl. Serum levels of methylmalonic acid, homocysteine, or both, were increased in 98.5% of patients.
Vitamin B₁₂ deficiency is the primary cause of megaloblastic anaemia in Zimbabwe and, contrary to textbook statements, is often due to pernicious anaemia. Isolated folate deficiency is less common. As reported in industrialized countries 75 years ago, anaemia is almost always present and often severe. Neurological dysfunction due to vitamin B₁₂ deficiency is most prominent in patients with mild to moderate anaemia.     

Increased Agglutinability by Anti-i of Red Cells in Sideroblastic and Megaloblastic Anaemia

Agglutinin titres have been performed using anti-I and anti-i cold antibodies on red cells from patients with sideroblastic, iron deficiency and megaloblastic anaemias, as well as on normal adult and cord blood red cells.
Raised i antigen titres were found using the red cells of 13 of 15 patients with sideroblastic anaemia and of seven of eight patients with megaloblastic anaemia but in none of eight patients with iron deficiency anaemia and in one of 17 patients with megaloblastic anaemia in remission. Those patients who had an elevated i antigen titre usually also had an elevated I antigen titre.
The raised i and I antigen titres of the red cells of a patient with pernicious anaemia were shown to decrease markedly within 3 months of the onset of B₁₂ therapy, indicating that the alteration probably persists for the life-span of the red cell but that the cause of the alteration is reversible once the underlying blood disorder is corrected.
It is believed that disordered erythropoiesis results in membrane alterations which lead to the increased agglutinability, but the cause is not known. It does not appear that a shortened red cell maturation time is a necessary factor.     

巨幼细胞性贫血患者骨髓检查结果分析

目的：了解巨幼细胞性贫血患者骨髓及实验室检查结果。方法：对222例巨幼细胞性贫血患者的骨髓象及血液的红细胞、白细胞、血小板和血清叶酸、维生素B12等结果进行总结分析。结果：有129例（58.1%）表现为全血细胞减少,以叶酸缺乏为主185例（83.3%）。叶酸、维生素B12缺乏越严重,骨髓中巨变等病态造血越明显。结论：巨幼细胞性贫血患者骨髓造血病态的程度往往反映患者营养不良的程度。     

A case of macrocytosis and megaloblastic erythropoiesis of unknown aetiology

The clinical and laboratory features in a non-anaemic 23-year-old female with marked macrocytosis and florid megaloblastic erythropoiesis of unknown aetiology are described. The bone marrow cells gave a normal deoxyuridine-suppressed value indicating that the megaloblastic erythropoiesis was not caused by vitamin B12 or folate deficiency or an impairment of the methylation of deoxyuridylate due to any other cause. The megaloblastic changes were associated with a marked degree of dyserythropoiesis, the most frequent ultrastructural abnormality being the presence of single or multiple intranuclear clefts. The characteristic light and electron microscope features of type I and type III congenital dyserythropoietic anaemia, in which mild megaloblastic changes may be seen, were absent. The distribution of the erythroblasts in the cell cycle was grossly abnormal and similar to that in severe pernicious anaemia; a high proportion of the cells were in the G2 phase and a substantial proportion seemed to have become arrested after progressing through part of the S phase. The bone marrow macrophages contained phagocytosed erythroblasts indicating that erythropoiesis was ineffective. It seems likely that the primary biochemical defect in the erythroblasts was some congenital disorder of DNA or nucleoprotein synthesis.     

Significance of Large Red Blood Cells

S ummary . Two observers examining marrow films from 81 patients for the presence of normoblastic and megaloblastic haemopoiesis obtained good agreement when the underlying disorder gave rise to definite vitamin B₁₂ or folate deficiency confirmed by microbiological assay. When microbiological assay of serum vitamin B₁₂ and red cell folate excluded vitamin B₁₂ or folate deficiency, the marrow changes were often too minor in degree for decisive diagnosis despite the presence in many cases of large red cells.
The normal mean corpuscular volume (MCV) set in relation to a PCV excluding trapped plasma was 80–90 fl. The MCV of red cells was often elevated above 90 fl in patients without a megaloblastic anaemia and in this series was often above 100 fl in patients with aplastic anaemia, sideroblastic anaemia, myxoedema, and neoplasia. The larger the red cells, however, the more probable was the presence of megaloblastic haemopoiesis in the marrow.     

Pattern of haematological diseases diagnosed by bone marrow examination in Yemen: a developing country experience

There is lack of information about the relative prevalence of haematological disorders in Yemen and other Middle East countries. The aim of this study was to evaluate the pattern of haematological diseases diagnosed by bone marrow examination in Yemen considering the limited diagnostic facilities. At the referral haematology centre in Yemen, between November 1999 and November 2005, 785 patients >14 years old were evaluated by bone marrow examination. Relevant investigations were performed when needed. A total of 627 patients had haematological disorders other than lymphoma, and their data were analysed. There were 273 females and 354 males. A total of 159 patients had Acute myeloid leukaemia, 75 had acute lymphocytic leukaemia, 87 had chronic myeloid leukaemia, 36 chronic lymphocytic leukaemia, eight had multiple myeloma, 13 myelodysplastic syndromes, seven myelofibrosis, seven polycythaemia vera, three primary thrombocythaemia, two hairy cell leukaemia, two metastases, 36 aplastic anaemia, 29 immune thrombocytopenic purpura (ITP), nine autoimmune haemolytic anaemia, three pernicious anaemia, 65 iron deficiency anaemia, 57 megaloblastic anaemia and malaria, 18 mixed deficiencies, and 11 patients had visceral leishmaniasis. Sex- and age-related distribution of the various disorders was also presented. In conclusion, the leukaemias were the most frequently encountered diagnosis followed by iron deficiency anaemia, megaloblastic anaemia and malaria, aplastic anaemia and ITP respectively. The other haematological disorders were less common. These findings are comparable with that seen in other developing and developed countries.     

Lymphocyte subpopulations in megaloblastic anaemia due to vitamin B-12 deficiency

Lymphocyte subpopulations were measured in the blood of 17 patients with megaloblastic anaemia due to vitamin B-12 deficiency. 14 patients had pernicious anaemia and 3 others were gastrectomized. By using monoclonal antibodies recognizing T cell surface markers and immunofluorescence microscopy, we found a significant decrease in the number of circulating suppressor T cells and an increase in the ratio of helper to suppressor T lymphocytes in pernicious anaemia patients. This finding may be related to other immune abnormalities found in pernicious anaemia, e.g. the presence of multiple autoantibodies.