Enzymatic Diagnosis of Megaloblastic Anaemia

S ummary A comparison of the lactate dehydrogenase (LDH) isoenzyme pattern found in the red cells from patients with megaloblastic anaemia, with that of normal human red cells, and the red cells from patients with iron deficiency and haemolytic anaemias, has shown that megaloblastic anaemia, whether due to vitamin B₁₂ deficiency, folate deficiency, or a mixed deficiency of both vitamins, causes a characteristic reversal in distribution of the faster moving isoenzymes LDH-1 and LDH-2. In normal red cells and those present in other anaemias, LDH-2 activity is always greater than LDH-1 activity, but in all cases of megaloblastic anaemias examined, LDH-1 was consistently greater than LDH-2 activity. This reversed LDH isoenzyme pattern is also present in the serum in megaloblastic anaemia, and is almost certainly derived from intramedullary destruction of megaloblastic precursor cells, with very little contribution from the circulatory red cells. It is well known that a feature of megaloblastic anaemia is a raised serum LDH activity, and it has been shown that a simple chloroform inhibition test made on either serum showing a raised serum LDH of over 900 units, or on the red cells, will give 2 good indication that this reversed pattern is present.     

Haematological Effects of the Idiopathic Splenomegaly Seen in Uganda

The haematological effects of idiopathic splenomegaly have been studied in a group of 15 Ugandan patients. The results of the main investigations were compared with those obtained in five patients with miscellaneous diseases and without palpable splenomegaly.
The splenic enlargement was mainly due to reticuloendothelial hyperplasia and not to sinusoidal congestion.
A normochromic anaemia was present in almost all the patients and a proportion had leucopenia and thrombocytopenia. One patient presented with a crisis of anaemia; she was later shown to have red cell G6PD deficiency which was believed to be coincidental.
Three aetiological factors were found in relation to the anaemia; reduction of red cell survival time, present in all the patients, haemodilution from expansion of the plasma volume, and the exclusion of a proportion of the red cell mass from the general circulation by the spleen. Six to 39 per cent of the red cell volume, the amount varying with spleen size, was sequestered in a slow mixing compartment, believed to be the extrasinusoidal spaces (the spleen pool) in the patients with splenomegaly. No spleen pool could be shown in any of the patients without splenomegaly.
Intrasplenic red cell destruction was not conspicuous and it has been suggested that the red cells, after damage from repeated stagnation in the spleen pool, are destroyed widely throughout the body.
The reasons for expansion of the plasma volume is not known. It relates to spleen size but cannot wholly be explained by plasma in the spleen pool.
A good result from splenectomy was obtained in six out of seven patients in whom this was undertaken. Two months after operation the red cell survival time was corrected in the patients in whom this was measured. The rise in the PCV could be explained only by substantial reduction in plasma volume.     

Erythropoiesis, Red-Cell Creatine and Plasma Aldolase Activity in Anaemia in the Rabbit and Man

S ummary . It has been shown that in the rabbit plasma aldolase was not increased by phlebotomy whereas in haemolytic anaemia produced by acctylphenylhydrazine there was a marked rise in plasma aldolase which was not, however, sustained throughout the period of falling haemoglobin. It is suggested that because of this dissociation of enzyme activity and haemoglobin the increased plasma aldolase activity in haeniolytic anaemia is not due to release of enzyme from the haemolysed red cells but that it is derived from immature precursor cells destroyed during transient ineffective erythropoiesis in the bone marrow.
The data obtained from these animal experiments have been used to elucidate the cause of the raised plasma aldolase which is a feature of some types of human anaemia. In iron-deficiency anaemia the plasma aldolase was not increased. In megaloblastic anacinia the plasma aldolase was increased in some but not in all patients. Evidence is adduced to support the hypothesis that the increase in plasma aldolase activity, when it is present, is due to release of enzyme from precursor cells destroyed in the marrow during ineffective erythropoiesis.
The creatine content of the red cell has been used as an index of the mean age of the cells in the peripheral circulation. Increased red-cell creatine in iron-deficiency anaemia is considered to be the result of a shortened survival of the cells in this condition.     

Inhibition of hexose monophosphate shunt in young erythrocytes by pyrimidine nucleotides in hereditary pyrimidine 5' nucleotidase deficiency

Recent reports have suggested that haemolytic anaemia in pyrimidine 5' nucleotidase (P5'N) deficiency might be due to impaired erythrocyte hexose monophosphate shunt (HMS). To investigate the relationship between pyrimidine accumulation, HMS impairment and shortened red-cell survival, we tested glucose 6-phosphate dehydrogenase (G-6PD), HMS, P5'N activities and the UV spectrum in whole red cells and in red cells of different age from 2 P5'N-deficient patients with different degrees of haemolytic anaemia. In whole red cells we found a reduction of both G-6PD and stimulated HMS activity in the presence of a variable amount of pyrimidine nucleotides (37.79 and 17.88 mumol/gHb respectively). A drastic inhibition of stimulated HMS activity was already present in the lightest red-cell fractions from patient 1, who presented a more severe haemolytic anaemia. The variable degree of pyrimidines found among red cell fractions, with a minor accumulation in the older red cells, supports the hypothesis that pyrimidine accumulation and HMS impairment occur in the younger erythrocytes of P5'N-deficient patients.     

Red Cell Life Span in Iron Deficiency Anaemia

Ashby survival studies were performed with hypochromic red cells of six patients with iron deficiency anaemia in the circulation of normal subjects and with normal red cells in two patients with iron deficiency anaemia. Three of the anaemic donors were professional donors with anaemia due to excessive blood donations as the only abnormality.
The life span of the hypochromic cells was decreased (46–85 days) whereas the normal cells survived normally or near normally in the patients with iron deficiency anaemia. Simultaneous ⁵¹Cr survival studies made with the hypochromic cells gave normal results in one instance and abnormal results in three. These findings suggest that hypochromic cells have a shortened life span due to an intracorpuscular defect.
In a complementary study, it was observed that hypochromic cells showing a decreased survival in two normal individuals, had a normal survival in a splenectomized subject.     

Hereditary sideroblastic anaemia and autosomal inheritance of erythrocyte dimorphism in a Dutch family

Size distribution curves of red blood cells were used to detect the presence of microcytes in peripheral blood of members of a Dutch family with hereditary sideroblastic anaemia. 22 of 49 members of this family have a bimodal erythrocyte volume distribution curve and a dimorphic blood picture. The pattern of inheritance of this morphological abnormality is clearly autosomal. It is suggested that the study of red blood cell size distribution curves may add valuable information on the pattern of inheritance in other families with hereditary sideroblastic anaemia.     

Lysis by Anti-I in Dyserythropoietic Anaemias: Role of Increased Uptake of Antibody

S ummary . Antibody lysis has been studied in PNH, in congenital dyserythropoietic anaemia and in other blood diseases with abnormal erythropoiesis. This last group of acquired dyserythropoietic anaemias included, especially, aplastic anaemia, megaloblastic anaemias, myelosclerosis and leukaemia. Cold-antibody lysis, complement sensitivity and anti-I antibody uptake by red cells were measured.
In both congenital dyserythropoietic anaemia and the acquired dyserythropoietic anaemias of all types there was increased cold-antibody lysis which was mainly or entirely due to increased antibody binding, without increased complement sensitivity. In PNH, on the other hand, there was slightly increased antibody uptake but lysis was much greater than could be accounted for by this alone and was mainly due to increased complement sensitivity. PNH thus appears to be uniquely different from the dyserythropoietic anaemias. Also, whereas in PNH complement sensitivity curves indicated the presence of two populations of red cells, in the dyserythropoietic anaemias the curves suggested that only one cell population was present.     

Splenic Red Cell Pooling in Hairy Cell Leukaemia

The splenic red cell volume has been measured directly by an isotope method with quantitative scanning in 10 patients with leukaemic reticuloendotheliosis (hairy cell leukaemia). The volume ranged between 211 and 726 ml (mean 410 ml, SD 158) and this constituted 15–48% (mean 28.1%, SD 9.5) of the total circulating red cell volume. This is an exceptionally large pool when compared with that found in myeloproliferative and lymphoproliferative disorders with the same degree of splenomegaly. It is consistent with the histological features which show marked red cell accumulation in the splenic cord areas. The red cell pooling in the spleen thus appears to be a significant factor in the anaemia and there was fairly good correlation between the percentage of improvement in the anaemia and the percentage of red cell volume contained in the spleen. By direct measurement of the splenic red cell pool, it is possible to predict the extent to which splenectomy will benefit the anaemia and this may also provide an indirect measure of the extent of bone marrow dysfunction in the causation of the anaemia.     

Red cell Th activation: biochemical studies

The peanut agglutinin from Arachis hypogea is a lectin that reacts with red blood cells expressing the Th antigen. The Th antigen has been said to be qualitatively similar to the T antigen, a well-defined antigen due to desialylation of glycophorin A and B that also reacts with the peanut agglutinin. We examined Th activated red blood cells from two patients with Fanconi's anaemia using 125I radiolabelled peanut agglutinin as a probe in Western blotting of red blood cell membrane proteins. We also probed the surface of intact Th activated red blood cells for structures related to the T antigen using [3H]sialic acid and a purified sialyltransferase. Neither of these techniques found antigens on the Th activated red blood cells that were similar to the antigen found on T activated red blood cells. These results show that the Th antigen in Fanconi's anaemia is qualitatively different to the antigen found in T activation.     

Studies of Splenic Function in the Myeloproliferative Disorders and Generalized Malignant Lymphomas

The relative importance of splenic red-cell pooling, sequestration and cell destruction in the causation of anaemia has been studied in 29 patients—16 with generalized lymphoproliferative disease, 12 with myeloproliferative disease and one with idiopathic autoimmune haemolytic anaemia.
A scanning method with [¹¹C]carbon monoxide was used for direct in vivo measurement of splenic red-cell volume, and the spleen was delineated by-a scan after injection of ⁸¹Rb-labelled red cells, damaged with non-radioactive 1-mercuri-2-hydroxypropane (MHP). The clearance time of the damaged cells from the circulation was used as an index of splenic function. The fraction of red cells in the spleen varied from 2.9% to 32% and the splenic red-cell volume ranged from 38 to 1000 ml. In patients with lymphoproliferative disorders their spleens contained a smaller proportion of red cells, relative to splenic size, than patients with myeloproliferative disease. Clearance of cells damaged with 1-mercuri-2-hydroxypropane (MHP) was 30–60 min in normal subjects. Slow clearances were found in some patients with lymphosarcoma; fastest clearances occurred in patients with obvious haemolytic anaemia. No clear relationship was noted between the rate of clearance and splenic size or splenic red-cell volume.     

The erythrocyte effects of haemoglobin O(ARAB)

To test the hypothesis that HbOARAB induces an increase in red cell mean corpuscular haemoglobin concentration (MCHC), we studied members of four Tunisian families who were either homo- or heterozygous for HbOARAB or were double heterozygotes for HbS and HbOARAB. The alpha-gene status was also tested. The findings included: (1) Distinctive variation in red cell density (MCHC) as determined by separation of red cells on isopycnic gradients: (a) All red cells from patients homozygous for HbOARAB were denser than normal red cells, as is observed for homozygous HbC patients. (b) In patients heterozygous for HbOARAB, red cell density was strongly influenced by the presence of alpha-thalassaemia. The coexistence of -alpha/alphaalpha resulted in an average red cell density slightly greater than normal (AA) red cells. Patients heterozygous for HbOARAB with a normal complement of four alpha genes had denser red cells similar to sickle cell disease with some cells of normal density but with most cells very dense. (c) Finally, the double heterozygotes for HbS and HbOARAB had significant haemolytic anaemia and red cells denser than normal with some as dense as the densest cells found in sickle cell anaemia. (2) Reticulocytes in patients homozygous for HbOARAB were found in the densest density fraction of whole blood. (3) Cation transport in patients homozygous for HbOARAB was abnormal, with K:Cl cotransport activity similar to that of HbS-Oman and only somewhat lower than in sickle cell anaemia red cells. The activity of the Gardos channel was indistinguishable from that found in HbS, HbC and HbS-Oman cells. We conclude that the erythrocytic pathogenesis of HbOARAB involves the dehydration of red cells due, at least in part, to the K:Cl cotransport system. The similarity of the charge and consequences of the presence of both HbC and HbOARAB, which are the products of mutations at opposite ends of the beta-chain, raises the possibility that this pathology is the result of a charge-dependent interaction of these haemoglobins with the red cell membrane and/or its cytoskeleton and that this abnormality is present early in red cell development.     

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.     

Microangiopathic haemolytic anaemia and thrombocytopenia following lung volume reduction surgery in a single lung transplant recipient on maintenance tacrolimus (FK506) therapy

Microangiopathic haemolytic anaemia (MAHA) describes intravascular haemolysis due to mechanical destruction of red cells as a result of pathological changes in small blood vessels. It is well recognized as a complication of cyclosporin A therapy in solid organ transplantation but has been uncommonly reported in association with tacrolimus therapy and never before in the setting of lung transplantation. Discussed is a 54-year-old female recipient of a left single lung transplant who developed anaemia, thrombocytopenia and red blood cell fragmentation consistent with MAHA following lung volume reduction surgery (VRS) of the native right lung in the setting of high serum tacrolimus levels. Treatment with fresh frozen plasma and plasmapharesis plus supportive therapy with blood and platelet transfusions resulted in successful resolution of the haemolytic process. Cyclosporin A was substituted for tacrolimus and 18 months later there has been no evidence of recurrence. Tacrolimus therapy is a rare cause of MAHA in solid organ transplants but the diagnosis should be considered if there is an unexplained fall in haemoglobin and/or platelet count in the context of high serum tacrolimus levels.     

Overproduction of structurally normal enzyme in man: hereditary haemolytic anaemia with increased red cell adenosine deaminase activity

The mechanism of red cell adenosine deaminase (ADA) accumulation in a case of hereditary haemolytic anaemia due to increased red cell ADA activity was investigated. ADA activity of the younger cells was twice that of the older cells. Rate of ADA synthesis in erythroid colony cells cultured from the patient's bone marrow cells was 11-fold greater than that from the normal. The accumulation of ADA in the patient seems to be due to the increased synthesis in precursors of red cells in spite of the increased degradation in peripheral blood.     

The Haematological Effects of Glucose-6-Phosphate Dehydrogenase Deficiency and Thalassaemia Trait: Interaction between the Two Genes at the Phenotype Level

The haematological effects of glucose-6-phosphate dehydrogenase (G6PD) deficiency and thalassaemia trait were evaluated in a field study of 317 individuals in an isolated Sardinian village, where both traits were present at high frequency. G6PD deficiency was diagnosed with rigid genetic criteria. Thalassaemia trait was diagnosed on the basis of abnormal osmotic fragility.
Complete G6PD deficiency resulted in mild anaemia with macrocytosis, a consequence of mild chronic haemolysis. Partial G6PD deficiency had a similar, but less marked effect. Thalassaemia trait resulted in mild anaemia, with marked microcytosis and moderate hypochromia.
The haematological effects of the combination of both traits were equal to the sum of the independent effect of each. The G6PD activity of the individual red cells was similar in thalassaemic and normal individuals. Because of the microcytosis, the G6PD activity per gram of haemoglobin per unit volume of red cells or of whole blood appeared to be elevated in thalassaemia trait.     

Use of erythropoietin in emergencies: massive intoxication by chloramines

Recombinant human Erythropoietin (rHuEPO) is normally used to correct anaemia in patients with End Stage Renal Disease (ESRD), that are in Regular Dialysis Treatment (RDT). This anaemia is usually due to the existence of two factors: A decrease in the erythropoiesis of the bone marrow and an increase in peripheral haemolysis and, consequently, a decrease in the life span of the red cells.     

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.     

Precautions surrounding blood transfusion in autoimmune haemolytic anaemias are overestimated

Background

It is very evident that many precautions are taken regarding transfusion of red blood cells in patients with autoimmune haemolytic anaemia. Frequently, considerable efforts are made to examine the indication and serological compatibility prior to transfusion in such patients. However, at times, this may unnecessarily jeopardize patients who urgently require a red blood cell transfusion.

Materials and methods

Thirty-six patients with warm-type autoimmune haemolytic anaemia were included in this study. All patients had reactive serum autoantibodies and required blood transfusion. Standard serological assays were employed for the detection and characterization of antibodies to red blood cells.

Results

A positive direct antiglobulin test was observed in all 36 patients, in addition to detectable antibodies in both the eluate and serum. Significant alloantibodies were detected in the serum samples of three patients (anti-c, anti-JK^a, and anti-E). In 32 patients, red blood cell transfusion was administered with no significant haemolytic transfusion reactions due to auto- and/or allo-antibodies. Due to overestimation of positive cross-matches three patients received no transfusion or delayed transfusion and died, and one patient died due to unrecognised blood loss and anaemia which was attributed to an ineffective red blood cell transfusion.

Discussion

Many of the reported recommendations regarding transfusion of red blood cells in autoimmune haemolytic anaemia are highly questionable, and positive serological cross-matches should not result in a delay or refusal of necessary blood transfusions.     

Iron Metabolism and Absorption Studies in the X-linked Anaemia of Mice

S ummary . The X-linked anaemia of mice (gene symbol, sla ) is hypochromic and microcytic, and the stainable iron stores are reduced. Chemical estimates of total body iron content and serum iron concentration show low values and the total serum iron binding capacity is elevated in anaemic mice. Rapid plasma iron clearance and increased iron utilization provide further confirmation of iron deficiency in anaemic animals. Alterations in activity of haem-containing enzymes have been sought in the heart, liver and kidney of anaemic mice, and slightly decreased activity found only in kidney cytochrome oxidase.
The blood volume of mice with X-linked anaemia is increased in spite of decreased red cell mass, and thus the anaemia is in part due to dilution. The cause of the increased blood volume has not been elucidated, but it may be related to splenic enlargement. In contrast to iron depleted humans and experimental animals, mice with X-linked anaemia show impaired rather than increased intestinal iron absorption, indicating that the anaemia is a consequence of iron malabsorption. The defect in iron absorption may be an isolated one, since evidence of impaired retention of orally administered radio-iodinated triolein, radio-zinc, radio-copper and radio-cobalt has not been found. It is suggested that the genetically-determined defect in the intestinal mucosa of mice with X-linked anaemia may be due to deficiency of either an enzyme or a carrier substance necessary for the normal transfer of iron from the intestinal mucosal cell to the plasma.     

Sites of red cell destruction in the anaemia of experimental burns

Summary . There are mainly two sites for red-cell destruction following thermal burn injury in albino rats. Intravascular haemolysis as well as hepatic destruction of red cells account for the elimination of red cells from the circulation during anaemia observed as early as 24 hours following burns. The anaemia that occurs on the 6th day of burns is associated mainly with splenic sequestration of red cells. Prednisolone was found to be ineffective in inhibiting the splenic sequestration of red cells, but splenectomy almost completely prevents the progression of anaemia.