Physical Address
304 North Cardinal St.
Dorchester Center, MA 02124
Investigation of Megaloblastic Anaemia: Cobalamin, Folate and Metabolite Status
Acknowledgements
Many thanks indeed to Dr Malcolm Hamilton and Mrs. Sheena Blackmore, authors of this chapter in editions 10 and 11 of this series. The descriptions of metabolic pathways for cobalamin, folate and homocysteine were assisted by Hematology Basic Principles and Practice and Homocysteine in Health and Disease. Thank you Annie Lee, Julie Bonser, staff at UK NEQAS Haematinics Department of Haematology and Good Hope NHS Trust. Thanks to Dr Agata Sobczynska-Malefora, Renata Gorska, David Card, Denise Oblein and the Nutristasis Unit at Guy’s and St. Thomas’ Hospital NHS Foundation Trust for ongoing expertise in the development and application of assays to establish vitamin status.
Cobalamin absorption and metabolism
Vitamin B 12 (cobalamin, B 12 ) belongs to a group of compounds named corrinoids. It is composed of a corrin ring and a central cobalt atom that is bound to two ligands. The lower ligand consists of a benzimidazole group attached to the corrin ring through a ribose-phosphate group. To confer metabolic utility in humans the upper ligand must consist of either a methyl or 5′-deoxyadenosyl moiety.
Vitamin B 12 is synthesised by microorganisms and enters the diet with food of animal origin. Although some edible green laver ( Enteromorpha spp.) and purple laver ( Porphyra spp.) contain substantial amounts of vitamin B 12 (≈ 63.6 μg/100 g dry weight and 32.3 μg/100 g dry weight respectively), higher plants do not require the vitamin for any function and have no mechanism for its production or storage.
In man, vitamin B 12 is required as a coenzyme for two reactions. One of the reactions occurs in the cytosol and requires methylcobalamin as a cofactor for methionine synthase during the remethylation of methionine from homocysteine. The remethylation of cobalamin requires the donation of the methyl group from 5′-methyltetrahydrofolate as it is converted to tetrahydrofolate, thus linking cobalamin to folate and 1-carbon metabolism. The second cobalamin-dependent reaction requires adenosylcobalamin and occurs in mitochondria. Adenosylcobalamin is a cofactor for the enzyme methylmalonyl-CoA mutase, which converts methylmalonyl-CoA to succinyl-CoA. The UK government recommends a daily intake of 1.5 μg of vitamin B 12 , with the European Union recommending 1 μg and the United States recommending 2.4 μg.
Methionine produced in the methylcobalamin-dependent reaction is converted to adenosylmethionine and is a vital source of methyl groups critical for a series of methylation reactions involving proteins, phospholipids, neurotransmitters, ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). Biochemical consequences of metabolic cobalamin insufficiency include an increase in the circulatory concentration of homocysteine and methylmalonic acid ( Fig. 10-1 ). Pathological consequences of a deficiency state include megaloblastic anaemia and neuropathies. Vitamin B 12 deficiency can cause lesions in the spinal cord, peripheral nerves and cerebrum. The most common symptoms are sensory disturbances in the extremities, memory loss, dementia and psychosis.
With the exception of those who consume a restricted diet (e.g. vegans and vegetarians), dietary intake of vitamin B 12 greatly exceeds metabolic requirement. The typical Western diet contains ≈ 4–6 μg/day of the vitamin, of which 1–5 μg is absorbed. Although the bioavailability of vitamin B 12 is considered high, uptake from a single serving decreases drastically once the 1.5–2.5 μg capacity of ileal receptors for the vitamin has been exceeded. A second dose of vitamin B 12 given 4–6 h later is once again absorbed with maximum efficacy. Body stores typically contain 1–5 mg of vitamin B 12 so that deficiency states may not develop until several years after the metabolic requirement has consistently exceeded dietary vitamin B 12 intake and absorption.
Vitamin B 12 deficiency is relatively common, with significant and variable clinical sequelae. , Causes of deficiency are shown in Table 10-1 . Estimates of the prevalence of vitamin B 12 deficiency are dependent on the criteria used to define a deficient state. Using serum vitamin B 12 of 200 pmol/l as a diagnostic cut-off, 3.9% of subjects over 60 years of age in the 2001–2004 National Health and Nutrition Examination Survey in the USA were defined as deficient. Prevalence varied by ethnic group with 4.1, 2.0 and 1.7% of non-Hispanic white, non-Hispanic black and Mexican Americans respectively being defined as deficient. Using serum vitamin B 12 < 147 pmol/l and homocysteine > 20 μmol/l, the prevalence of vitamin B 12 deficiency was ≈ 5% in people 65–74 years of age, and more than 10% in people 75 years of age or older. Using holotranscobalamin (HoloTC) and methylmalonic acid in tandem, 11% of a hospital patient population in the UK were defined as deficient.
Table 10-1
The causes of vitamin B 12 deficiency
| Defect | Cause | Supportive Information/Diagnostic Tests |
|---|---|---|
| Decreased intake | Malnutrition Reduced intake of animal products Strict vegan diet Breastfed babies of mothers who are vegetarian or B 12 deficient Poor dietary intake in elderly |
Dietary history Ethnic origin/culture |
| Impaired gastric absorption | Atrophic gastritis with achlorhydria Gastrectomy – partial or total Zollinger–Ellison syndrome |
Endoscopic and gastric biopsy findings History of gastric surgery Multiple gastric and duodenal ulcers Pancreatic adenoma on imaging |
| Addisonian pernicious anaemia | Diagnostic criteria for pernicious anaemia (see Table 10-4 ) | |
| Failure of trypsin release of B 12 from R binding proteins | Pancreatic insufficiency | Pancreatic function tests; exocrine pancreatic dysfunction results in reduced absorption but clinical deficiency is rare |
| Impaired intestinal absorption as a result of failure of B 12 -intrinsic factor complex uptake in ileum | Ileal resection or disease e.g. Crohn disease Inflammatory bowel disease and tuberculous ileitis Tropical sprue Blind loop syndrome |
Radiological, enteroscopic or capsule camera study of small bowel for Crohn disease of terminal ileum or tuberculous ileitis Small bowel biopsy Radiolabelled lactose breath tests for bacterial overgrowth. Absorption returns to normal after antibiotic therapy |
| Congenital/inherited | Intrinsic factor receptor deficiency/defect Imerslund–Gräsback syndrome Congenital deficiency of intrinsic factor – ‘juvenile’ pernicious anaemia Inborn errors of cobalamin metabolism |
Subjects of Scandinavian origin Serum and urinary methylmalonic acid and metabolite measurement |
| Abnormal transport proteins | Haptocorrin deficiency
Transcobalamin deficiency Possible fall in holotranscobalamin levels in elderly |
No evidence of clinical deficiency but low serum cobalamin levelsMegaloblastic anaemia in presence of normal cobalamin levels; transcobalamin and holotranscobalamin levels reduced |
| Excess consumption | Haemolysis HIV infection |
|
| Acquired drug effects | Alcohol: impedes absorption as consequence of gastritis Nitrous oxide: irreversibly binds to cobalt atom in B 12 and deactivates it Proton pump inhibitors: reduce gastric acid production H 2 receptor antagonists: reduce gastric acid production Metformin: impedes absorption Colchicine: reduces IF-B 12 receptors Slow K: impedes absorption Cholestyramine: decreases gastric absorption |
Chronic repeated exposure |
HIV, human immunodeficiency virus; IF, intrinsic factor.
An overview of the steps involved in the absorption of vitamin B 12 is shown in Figure 10-2 . Ingested cobalamin is released from food proteins by pepsin and gastric acid. Two proteins then compete for the free cobalamin: a glycoprotein named intrinsic factor, which is made in gastric parietal cells, and haptocorrin (previously known as transcobalamin I and also referred to as R binder), which is produced by salivary glands. At acidic pH, intrinsic factor (IF) has a very low affinity whilst haptocorrin has a high affinity for vitamin B 12 . Thus vitamin B 12 binds to haptocorrin in the stomach. Haptocorrin primarily serves to protect vitamin B 12 from acid degradation in the stomach by producing a haptocorrin–vitamin B 12 complex. Metabolically inert cobinamides (an intermediate in porphyrin and chlorophyll metabolism) that are present in the diet are also bound. As the contents of the stomach enter the first part of the duodenum a relatively alkaline environment is encountered. The haptocorrin is partly digested by proteases secreted by the pancreas, which frees the vitamin, permitting it to attach to IF. The intrinsic factor–cobalamin complex attaches to cubam receptors, which consist of amnionless and cubilin, and the complex is taken up by endocytosis into the ileal cell. After internalisation, vitamin B 12 is freed and transported into the blood, possibly by an adenosine triphosphate (ATP)-dependent carrier, where it meets transcobalamin (previously known as transcobalamin II) and haptocorrin. Unsaturated transcobalamin is more abundant so most newly absorbed vitamin B 12 binds to it. Transcobalamin has a rapid turnover and is responsible for the daily transport of ≈ 4 nmol of vitamin B 12 into cells. Haptocorrin is almost fully saturated with vitamin B 12 (and inactive vitamin B 12 analogues) and carries the major part of the vitamin in the circulation. The metabolism of this protein, which attaches to cell surface receptors on liver and other storage cells, is slow, with a turnover of 0.1 nmol of vitamin B 12 daily.
There are receptors for holotranscobalamin (TC receptors) on the surface of every DNA-synthesising cell in the human body. At the cellular level in the target tissue, HoloTC undergoes endocytosis via the transmembrane TC receptor before lysosomal degradation, releasing cobalamin for metabolic reactions.
Cobalamin undergoes enterohepatic circulation via the liver and bile ducts with 1.4 μg/day excreted in the bile, of which 1 μg/day is reabsorbed in the ileum.
Folate absorption and metabolism
Folate is a generic term that refers to a group of water-soluble vitamins that function as cofactors by carrying and chemically activating single carbons to support biosynthetic pathways. Causes of folate deficiency are shown in Table 10-2 .
Table 10-2
The causes of folate deficiency
| Defect | Supportive Information/Diagnostic Tests |
|---|---|
| Reduced intake | |
| Poor diet, particularly alcoholics (wine and spirits because beer contains folate) Elderly or students (‘tea and toast diet’) Dietary fads Premature babies Unsupplemented parenteral nutrition |
Dietary and alcohol history |
| Malabsorption | |
|---|---|
| Coeliac disease (often with coexisting iron deficiency) Tropical sprue Small bowel resection, malabsorption syndromes |
Anti-endomysial, antigliadin tests, antitissue transglutaminase Small bowel biopsy |
| Drug effects | |
| Sulphasalazine, methotrexate, trimethoprim-sulphamethoxazole, pyrimethamine, phenytoin, sodium valproate, oral contraceptives | Drug history Bone marrow aspiration |
| Hereditary hyperhomocysteinaemia | |
| Homozygotes for C677T MTHFR may have lower total folate levels and/or proportionally lower availability of 5-methyl THF | |
| Increased folate turnover | |
| Pregnancy: progressive increase in requirement in third trimester Increased requirements for breastfeeding Skin disease – severe psoriasis or exfoliation Haemodialysis Haemolysis: haemoglobinopathy, paroxysmal nocturnal haemoglobinuria, autoimmune haemolytic anaemia (see Chapter 11 , Chapter 12 , Chapter 13 ) |
|
Folate polyglutamates are thermolabile and found in fruits and vegetables, in particular in leafy green vegetables. Before absorption can take place, dietary folate polyglutamates must be hydrolysed to monoglutamates by hydrolases, operating maximally at pH 5.5 in the presence of zinc and then rapidly converted to polyglutamates in cells. Folate carriers transport polyglutamates rapidly into the luminal cells where they are methylated using methylcobalamin as a cofactor and reduced to 5-methyltetrahydrofolate (5-methyl THF) in the enterocyte before entering the portal venous system. Unconverted polyglutamates remain in the luminal cells.
There is significant enterohepatic recirculation of folate, amounting to 90 μg/day. Biliary drainage results in a rapid fall in serum folate levels, whereas deprivation of dietary folate takes up to 3 weeks to cause a decrease in serum levels. Two-thirds of plasma folate is non-specifically bound to plasma folate-binding proteins including albumin, and one third circulates as free folate.
There is sufficient retention of folate by the renal tubules to prevent urinary folate loss; this is achieved by megalin uptake of filtered folate-binding protein and the bound folate. Cubam, which binds intrinsic factor-cobalamin complex, is also important in the uptake of albumin from the renal tubules, which may also contribute to folate retention.
Folate transport into cells is dependent upon two mechanisms: reduced folate carrier (58 kDa), which is a low-affinity high-capacity system; and reduced folate receptors (44 kDa), of which there are three isoforms – alpha and beta are attached to the cell surface through a glycosyl-phosphatidylinositol anchor and gamma is secreted by enteric mucosal cells. Methyltetrahydrofolate bound to the folate receptor undergoes endocytosis by clathrin-coated pits or caveolae. Passive diffusion is an alternative mechanism by which folate can enter cells. The relative contributions of the different mechanisms are not known. Folate receptors may be expressed on malignant cells and have become potential targets for delivery of cytotoxic agents linked to folate.
Folates participate in 1-carbon metabolism and thereby facilitate the essential cellular metabolism of methionine, serine, glycine, choline and histidine in the biosynthesis of purine and deoxythymidine monophosphate (dTMP) in the synthesis of pyrimidines and thus DNA ( Fig. 10-1 ).
Intracellular folates are compartmentalised between the cytosol and mitochondria, and the major forms are tetrahydrofolate (THF), 5-methyl THF and 10-formyl THF. Homocysteine is converted to methionine by methionine synthase using methylcobalamin as a cofactor and 5-methyl THF as the methyl group donor. Cobalamin deficiency therefore results in inactivation of methionine synthase, leading to an accumulation of 5-methyl THF, which cannot be converted back to 5,10-methylene THF. Folate is then unavailable for pyrimidine and purine synthesis – this is known as the methyl-trap hypothesis, which was advanced to explain why cobalamin deficiency often results in a functional folate deficiency. Furthermore, 5-methyl THF is a very poor substrate for the enzyme responsible for folate polyglutamation, folylpoly-γ-glutamate synthetase, which prefers THF and 10-formyl THF. Folate deficiency is thought to cause megaloblastic anaemia by inhibiting the production of 5,10-methylene THF polyglutamate, which acts as a cofactor in the rate-limiting step in the production of DNA, the synthesis of dTMP. Thus, in the absence of cobalamin, polyglutamate synthesis ceases and monoglutamate forms are not retained by cells. This explains why, in cobalamin deficiency, serum folate levels may be found to be elevated and red cell folate levels normal or low.
Haematological features of megaloblastic anaemia
Macrocytosis is the most common reason that vitamin B 12 status is investigated. Megaloblastic anaemia resulting from impaired DNA synthesis is characterised by the presence of megaloblastic red cell precursors in the bone marrow and occasionally also in the blood. Megaloblasts have a characteristic chromatin pattern ( Fig. 10-3 ) and increased cytoplasm as a result of asynchrony of nuclear and cytoplasmic maturation with a relatively immature nucleus for the degree of cytoplasmic haemoglobinisation. A delay in nuclear maturation caused by impaired DNA synthesis resulting from a lack of vitamin B 12 or folate is seen in all lineages, particularly granulocytic marrow precursors with giant metamyelocytes ( Fig. 10-4 ) and hyperlobated neutrophils with increased lobe size as well as an increased number of nuclear segments (see Fig. 5-10 ). In severe pernicious anaemia, a mean red cell volume (MCV) up to 130 fl occurs, with oval macrocytes, poikilocytes and hypersegmentation of neutrophils (> 5% with at least five nuclear lobes). The neutrophil hypersegmentation index is an equivalent automated parameter on some cell counters. The mean platelet volume is decreased, and there is increased platelet anisocytosis, as detected by the platelet distribution width (PDW). The MCV falls to 110–120 fl or even lower as megaloblastic change advances, as a result of the appearance of red cell fragments and small poikilocytes. Howell–Jolly bodies and basophilic stippling may be seen in the red cells.
Differential diagnosis of macrocytic anaemia
Macrocytic red cells are also seen in myelodysplastic syndromes and myelodysplastic/myeloproliferative neoplasms, which can be suspected from the presence of hypogranular neutrophils (see Fig. 5-76 ) or monocytosis. Excess alcohol consumption results in an increased MCV with round macrocytes, although rarely does the MCV exceed 110 fl unless coexisting folate deficiency is present. Hypothyroidism, liver disease, aplastic anaemia and the rare inherited orotic aciduria and Lesch–Nyhan syndromes also have a high MCV. Automated reticulocyte counts facilitate detection of increased red cell turnover and high MCV as a result of haemolysis or bleeding. Coexisting iron deficiency or thalassaemia trait may mask macrocytic changes, although a high red cell distribution width indicates anisocytosis and the need for blood film review. Congenital dyserythropoietic anaemias types I and III and erythroleukaemia exhibit some features of megaloblastic erythropoiesis that are unrelated to vitamin B 12 and folate. Drugs interfering with DNA synthesis result in macrocytosis and megaloblastic erythropoiesis ( Tables 10-1 and 10-2 ).
Metabolic insufficiency
No international consensus view on what constitutes vitamin B 12 deficiency has been reached, partly because harmonised laboratory diagnostic standards have yet to be defined. However, it is clear that the investigation of vitamin B 12 and folate status must not be restricted to individuals with classic features of megaloblastic anaemia because neuropathy, optic atrophy and ≈ 20% of neuropsychiatric changes occur in the absence of macrocytosis or anaemia. , Metabolic evidence of insufficiency states (i.e. elevations in homocysteine and methylmalonic acid concentration) are frequently identified in the laboratory without clinical signs or symptoms.
Currently ≈ 75 countries require fortification of wheat flour produced in industrial mills with folic acid. In the USA and Canada, dietary supplementation with folate was introduced in 1998 to reduce neural tube defects. Subsequent follow-up showed a 25–46% reduction in the incidence of neural tube defects.
Folate and/or vitamin B 12 insufficiency causes an elevated circulatory concentration of homocysteine as a result of reduction in methionine synthesis ( Fig. 10-1 ). Homocysteine is an independent risk factor for vascular disease , and is also associated with an increased risk of venous thrombosis. , Lowering homocysteine levels may reduce the incidence of myocardial infarction and stroke.
Testing strategy for suspected cobalamin or folate deficiency
The application of a suitable testing strategy for patients suspected of having cobalamin or folate deficiency is shown in Tables 10-3 and 10-4 . Table 10-3 lists the important laboratory investigations that should be performed. Table 10-4 provides a list of clinical and laboratory features for the diagnosis of pernicious anaemia. Table 10-5 highlights the important clinical details that should be elicited by the clinician and submitted with the request to assist the laboratory in interpretation.
Table 10-3
Laboratory tests in suspected cobalamin or folate deficiency
| Diagnostic Tests | Diagnostic Features Suggestive of Cobalamin or Folate Abnormality | Will Help to Exclude | Pitfalls |
|---|---|---|---|
| Full blood count | Macrocytosis | – | Macrocytosis and anaemia may be absent despite neuropathy |
| Blood film | Oval macrocytes, hypersegmented neutrophils (> 5% with ≥ 5 lobes); Howell–Jolly bodies suggests hyposplenism and therefore coeliac disease as a cause of the deficiency | Myelodysplastic syndromes (hypogranular or hypolobulated neutrophils, dimorphic red cells), alcohol excess/liver disease (round macrocytes, target cells, stomatocytes), haemolytic anaemia (see Chapter 5 ) | Hypersegmented neutrophils are not invariably present; they can also occur during cytotoxic therapy |
| Reticulocyte count | Absolute count low pretreatment | Reticulocyte response at day 6 post-therapy confirms response to B 12 or folate therapy provided only low dose is given | Reticulocyte response may be blunted if inadequate iron stores |
| Bone marrow aspirate (including Perls stain) before treatment or within 24 h of cobalamin or folate therapy – indicated if severe, unexplained macrocytic anaemia | Megaloblastic erythropoiesis, giant metamyelocytes, hypersegmented neutrophils, ring sideroblasts infrequent | Myelodysplastic syndromes, aplastic anaemia | Megaloblastic change is not necessarily a result of deficiency; can be drug induced or a feature of a myelodysplastic syndrome |
| Serum B 12 | B 12 < 180 ng/l suggestive of cobalamin deficiency, may be a result of pernicious anaemia, veganism or gastrectomy; in the absence of these causes, may result from malabsorption of protein-bound B 12 (e.g. as a result of achlorhydria) B 12 < 150 ng/l highly suggestive of cobalamin deficiency |
B 12 < 180 ng/l with no clinical signs or symptoms and normal MMA and homocysteine reflects poor specificity of total B 12 assay. B 12 levels may be borderline low due to severe folate deficiency; give folic acid and monitor B 12 level unless neurological abnormalities present | B 12 > 180 ng/l but presence of neuropathy or strong clinical suspicion of B 12 deficiency requires a therapeutic trial or additional tests, such as MMA, holotranscobalamin and homocysteine: if holotranscobalamin is low then treat with B 12 and monitor response by repeat metabolite levels at day 6; B 12 deficiency is confirmed by high MMA and homocysteine that fall on treatment |
| Serum folate | Low level, particularly if red cell folate also low confirms deficient state | Subject to diurnal variation. Low levels may result from recent deterioration in diet. Conversely low serum folate levels are rapidly corrected by improved diet. | |
| Red cell folate | Low level, particularly if B 12 deficiency is excluded | Low red cell folate and high serum folate occur in cobalamin deficiency – treat with B 12 | |
| Serum holotranscobalamin | Early marker of B 12 deficiency. Holotranscobalamin < 25 pmol/l highly suggestive of cobalamin deficiency. Often as low as 5 pmol/l in pernicious anaemia. | Holotranscobalamin 25–50 pmol/l suggests measure MMA (or homocysteine if MMA not available). | Subject to recent dietary change (within 24h). Particularly useful in pregnancy, where levels unaffected by trimester. |
| Elevation of serum holotranscobalamin levels at 24-48 h from baseline in response to oral 9 μg cyanocobalamin 6 hourly × 3 doses | Subjects with dietary deficiency show rapid elevation of HoloTC by > 10 pmol/l or > 22% increase from baseline. Pernicious anaemia shows enhanced holotranscobalamin levels after addition of oral recombinant intrinsic factor. | ||
| Intrinsic factor antibody test (test if serum B 12 is < 150 ng/l) | Positive in 50–60% of cases of pernicious anaemia and, when positive, obviates a B 12 absorption test | False positive (rare). Negative in 40–50% of cases of pernicious anaemia; if negative, proceed to B 12 absorption test | |
| Schilling test (part I, basic test; part II, with intrinsic factor; part III, following course of antibiotics; part IV, pancreatic enzymes taken for 3 days) | Part I < 5% and part II normal or near normal confirms malabsorption as a result of lack of intrinsic factor (e.g. pernicious anaemia). Parts I and II abnormal suggests malabsorption not resulting from intrinsic factor deficiency. * Part III abnormal indicates abnormal bacterial grown. Part IV abnormal indicates pancreatic insufficiency. | Reagents not currently available. Awaiting recombinant intrinsic factor supplier. Invalid in renal failure; Part II may not correct in pernicious anaemia if intrinsic factor antibodies are present at high concentration in gastric juice | |
| Upper gastrointestinal endoscopy and duodenal biopsy † | Villous atrophy in coeliac disease. Coeliac disease indicated by positive serological tests for endomysial antibodies (EMA) and tissue transglutaminase antibodies (tTGA) | ||
| Serum gastrin or gastric juice pH | Raised serum gastrin or gastric juice pH of > 6 confirms achlorhydria: if not present, diagnosis of pernicious anaemia is suspect | ||
| Serum MMA and plasma or serum homocysteine, before treatment or before and 6 days after treatment ‡ | Raised homocysteine in folate and B 12 deficiency; raised MMA in B 12 deficiency, which is helpful to confirm deficiency if B 12 is low and IF antibodies are absent. Correction of elevated metabolite levels after cobalamin therapy provides evidence of biochemical response. | Lack of significance of low B 12 is indicated by normal MMA and homocysteine and no clinical signs. Note serum homocysteine requires sample taken on ice and separated before any haemolysis. Alternatively serum can be collected into Kabevette sample collection tubes (www.kabe-labortechnik.de) and separated within 36 hrs. | Both MMA and homocysteine are elevated in renal impairment. MMA is often elevated in the elderly and cannot be used in isolation without either B 12 or HoloTC measurement. Homocysteine is not specific for cobalamin deficiency, being elevated in folate deficiency, in smokers and in hyperhomocysteinaemia. |
HoloTC, holotranscobalamin; IF, intrinsic factor; MMA, methylmalonic acid; MTHF, methyltetrahydrofolate.
* If Part II fails to correct, proceed to barium follow-through or small bowel enema to diagnose ileal disease (e.g. Crohn disease); Parts I and II abnormal, Part III normal indicates malabsorption resulting from bacterial overgrowth (e.g. blind loop syndrome).
† Upper gastrointestinal tract endoscopy is also useful if dyspepsia develops in known pernicious anaemia, to exclude gastric carcinoma.
‡ Plasma homocysteine assay is now widely available; in suspected B 12 deficiency it provides a useful test of biochemical cobalamin deficiency. Correction of elevated levels after treatment provides confirmation of deficiency.
Table 10-4
Clinical and laboratory checklist for diagnosis of pernicious anaemia
| Laboratory Criteria | Clinical Criteria | |
|---|---|---|
| Minor criteria | Macrocytosis, anaemia Raised plasma homocysteine Gastric pH > 6 Raised serum gastrin Positive gastric parietal cell antibody |
Parasthesiae, numbness or ataxia Hypothyroidism Vitiligo Family history of pernicious anaemia or hypothyroidism |
| Major criteria | Low serum B 12 (< 180 ng/l) or raised serum methylmalonic acid (> 0.75 μmol/l) in presence of normal renal function Megaloblastic anaemia not resulting from folate deficiency Positive intrinsic factor antibodies using high-specificity test Holotranscobalamin level < 25 pmol/l |
|
| Reference standard criteria | Schilling test * shows malabsorption of oral cyanocobalamin corrected by co-administration of intrinsic factor |
* Reagents for Schilling tests currently unavailable. A nonisotopic B 12 absorption test has been reported.
Table 10-5
Significance of clinical details in assessing possible deficiency of B 12 or folate
| Symptoms or Signs | Possible Significance |
|---|---|
| Tiredness, palpitations, pallor | Anaemia |
| Slight jaundice | Ineffective erythropoiesis |
| Neurological | |
| Cognitive impairment, optic atrophy, loss of vibration sense, joint position sense; plantar responses normal or abnormal; tendon reflexes depressed or increased | Cobalamin deficiency, subacute combined degeneration of the spinal cord and optic/sensory/motor peripheral neuropathies |
| Dietary and Gastrointestinal History | |
| Vegetarian or vegan; poor nutrition (e.g. ‘tea and toast diet’ in elderly or students); dietary fads Weight loss, bloating and steatorrhoea, particularly nocturnal bowel movements Mouth ulcers, abdominal pain, perianal ulcers, fistulae Glossitis, angular cheilosis and koilonychia Alcohol history |
Low iron stores and iron deficiency Cobalamin deficiency in babies born to mothers who are vegans Folate deficiency (often with iron deficiency) Features of malabsorption and folate deficiency, e.g. due to coeliac disease, tropical sprue Terminal ileal Crohn disease – cobalamin deficiency Cobalamin and combined iron deficiency Poor diet and interference with folate metabolism |
| History of Autoimmune Disease in Patient or Family | |
| Hypothyroidism, pernicious anaemia or coeliac disease | Increased likelihood of pernicious anaemia or coeliac disease |
| Surgery | |
| Gastrectomy/bowel resection | Cobalamin deficiency usually develops 2 years postgastrectomy Ileal disease resulting in cobalamin deficiency Blind loop syndromes |
| Physical Appearance | |
| Grey hair, blue eyes, vitiligo | Association with pernicious anaemia |
| Pregnancy | Increased iron and folate requirements. Cobalamin levels fall by 30% in the third trimester but without tissue deficiency Holotranscobalamin levels are unaltered in late pregnancy |
| Malabsorptive Syndrome | |
| Tropical sprue, bacterial overgrowth, fish tape worm in Scandinavian countries | Combined folate and iron deficiency Cobalamin deficiency |
| Drug History | See text |
| Other Haematological Disorders | |
| Myeloproliferative neoplasms, haemolytic anaemias, leukaemias Multiple myeloma |
Increased folate utilisation may result in folate deficiency Paraprotein interference with cobalamin assays resulting in falsely low cobalamin levels, which normalise on treatment of myeloma |
Utility of serum vitamin B 12 assays
Most commonly vitamin B 12 status is evaluated through the measurement of vitamin B 12 in serum with a move away from microbiological assays and radioisotope-dilution assays (both described in detail in earlier editions of this book) to the use of automated platforms based on competitive-binding luminescence technologies. Status is estimated from the abundance of vitamin B 12 in the circulation, and comparison against a predefined reference range. However, this approach gives no indication of vitamin B 12 utilisation at the tissue level. Serum vitamin B 12 assays generate a high rate of false-negative results. It has been estimated that up to 45% of vitamin B 12 -deficient subjects may be overlooked if only serum vitamin B 12 is used as a screening test. Studies have also shown poor positive predictive value (i.e. healthy persons with a low cobalamin level with no evidence of deficiency). ,
In particular, clinical utility is limited when the serum B 12 assay is applied to pregnant women, as there is an ≈ 50% decline in serum cobalamin at term when compared with nonpregnant women. This is caused both by haemodilution and by a decrease in concentration of circulating haptocorrin. A second limitation is that in rare cases patients lack haptocorrin and therefore have a low serum vitamin B 12 despite no signs of deficiency.
Although ≈ 80% of vitamin B 12 is carried by haptocorrin, extrahepatic cellular receptors for this form have not been identified. Circulatory levels of haptocorrin decline slowly in response to negative vitamin B 12 balance (i.e. when metabolic requirements exceed absorption of dietary vitamin B 12 ) and may take 3–6 years to fall below the lower limit of assay reference ranges. It is cobalamin present in the HoloTC form that is metabolically active. Adoption of HoloTC assays , , to determine the abundance of the physiologically active form of cobalamin is increasingly widespread in Australia, Austria, Canada, Germany, the Netherlands, Nordic countries, the UK and Switzerland. However, the mode of application and the assignment of cut-off values is variable (i.e. used as a sole status indicator with a suggested optimal cut-off of 32 pmol/l ; first line screening test in conjunction with a functional marker to evaluate indeterminate results ; or second line test used in tandem to support indeterminate results from serum B 12 measurement).
Sensitivity and specificity of cobalamin assays
Utility of receiver operator characteristic curves
Defining the sensitivity and specificity of current vitamin B 12 assays has been hampered by the difficulty in defining a truly deficient study population. Clarke et al . provided data that permit calculation of the specificity and sensitivity of current immunoassays for vitamin B 12 in the detection of cobalamin deficiency in a community study of 1621 subjects aged over 65 yr with normal renal function. Subjects were defined as cobalamin deficient if the methylmalonic acid was elevated above 0.75 μmol/l. Deficiency was found in 4.3% of subjects over 65 years of age with normal renal function. The mean vitamin B 12 level of these subjects was 151 pmol/l (202 ng/l) using the Siemens Centaur assay (range 110–199 pmol/l). Table 10-6 illustrates the calculation to derive specificity and sensitivity for the Siemens Centaur B 12 assay using a cut-off point of 200 pmol/l (270 ng/l).
Table 10-6
The calculation to derive specificity and sensitivity for the Siemens Centaur B 12 assay
| Community study of 1621 subjects with normal renal function in Banbury, UK | True cobalamin deficiency defined by methylmalonic acid of > 0.75 μmol/l with normal renal function | Absence of cobalamin deficiency defined by methylmalonic acid of < 0.75 μmol/l | |
| Siemens Centaur B 12 assay < 270 ng/l | 53 true positives (TP) | 437 false positives (FP) (27% of subjects studied) | Positive predictive value = TP/(TP + FP) = 53/490 = 13.2% |
| Siemens Centaur B 12 > 270 ng/l | 17 false negatives (FN) | 1114 true negatives (TN) | Negative predictive value = TN/(TN + FN) = 1114/1131 = 98.4% |
| 70 cobalamin-deficient individuals (4.3% of subjects studied). Sensitivity = TP/(TP + FN) = 53/70 = 75.7% | 1551 nondeficient. Specificity = TN/(TN + FP) = 1114/1551 = 71.8% |
False-positive rate (α) = FP/(FP + TN) = 437/1551 = 28.2% = 1 - specificity. False-negative rate (β) = FN/(TP + FN) = 17/70 = 24.3% = 1 − sensitivity.
This study demonstrates that, while values over 270 ng/l have a high (98.4%) negative predictive value for the presence of disease, values below 270 ng/l include a high percentage (28.2%) of individuals with normal methylmalonic acid levels and presumably no other evidence of cobalamin deficiency. This is reflected in the poor specificity (71.8%) of the assay using this cut-off point. The choice of the appropriateness of the cut-off point can be explored further using receiver operator characteristic (ROC) curves.
High vitamin B 12 levels have been described in subjects with no myeloproliferative neoplasm and who are not on cobalamin therapy or vitamin supplementation. This is thought to be due to immunoglobulin-complexed vitamin B 12 resulting in assay interference. False-normal vitamin B 12 levels , have been described in subjects with high-titre IF antibodies and may also occur due to the presence of heterophile antibody interference.
Utility of holotranscobalamin II assay
In the same study Clarke et al . demonstrated that the Axis-Shield/Abbott HoloTC assay has slightly superior ROC curves when compared with serum B 12 levels (see Fig. 10-5 ). The HoloTC assays gave a greater area under the curve, 0.85 versus 0.76 (for serum vitamin B 12 ) and superior sensitivity and specificity. In common with many tests used in the diagnostic laboratory the vitamin B 12 and HoloTC assays both generate a proportion of results that are considered to be indeterminate. This inherent limitation can be addressed through further secondary testing using a functional marker of status. Suitable functional markers include circulatory concentrations of methylmalonic acid and homocysteine. HoloTC has been shown to be unaffected by assay interference from high-titre IF antibodies. In addition, HoloTC is not subject to the ≈ 50% fall in total vitamin B 12 levels seen in normal pregnancy ( Fig. 10-6 ).
Utility of methylmalonic acid and homocysteine assays
In countries where the addition of folic acid to all enriched cereal-grain foods is not mandated, the utility of an elevation in plasma (total) homocysteine concentration to identify methionine synthase dysfunction in response to suboptimal vitamin B 12 availability is limited by a codependency on 5-methyl THF abundance. 5-Methyl THF is a more powerful nutritional determinant of homocysteine (≈ 3.5 times) than methylcobalamin. It is good practice to apply reference ranges for the interpretation of homocysteine that are age, sex and renal function specific. Examples of reference ranges suitable for use in the clinical setting are: ≤ 10 μmol/l, children < 15 yr; ≤ 13 μmol/l, females (during pregnancy < 10 μmol/l); ≤ 15 μmol/l, males aged 15–65 yr; ≤ 15 μmol/l, females; ≤ 17 μmol/l males aged 65–74 yr; ≤ 20 μmol/l all > 74 yr.
The interpretation of methylmalonic acid is considered the gold standard and the most representative marker of metabolic vitamin B 12 insufficiency. The four primary determinants of the serum concentration of methylmalonic acid are age, vitamin B 12 status, renal function and sex. The interpretation of methylmalonic acid results is therefore complex in elderly populations and those with impaired renal function. Examples of upper limits of the range are 280 nmol/l (< 65 years of age) and 360 nmol/l for patients over the age of 65.
Clinical and diagnostic pitfalls of folate assays
Serum folate is altered by acute dietary change and interruption of enterohepatic recycling; it can therefore be low without significant tissue deficiency. Red cell folate was originally advocated as correlating better with megaloblastic change , reflecting the mean folate status over the lifespan of the red cells (2–3 months), but a subsequent study suggested that little was to be gained by the addition of red cell folate analysis. Minor haemolysis in vitro may cause spurious elevation of serum folate levels because the red cell folate may be 10–20 times the serum value. More than half of patients with severe cobalamin deficiency have a low red cell folate because impaired methionine synthesis results in accumulation of 5-methyl THF monoglutamate, which diffuses out of cells resulting in a high serum folate. , Treatment with cobalamin alone will correct the low red cell folate and high serum folate levels. The interplay between serum vitamin B 12 , serum folate, red cell folate, plasma homocysteine and methylmalonic acid is shown in Table 10-7 . In the USA, some authors have advocated cessation of folate testing because folate deficiency has become very unusual since the introduction of dietary supplementation of flour. The causes of clinical deficiency and supportive information or diagnostic tests are shown in Table 10-2 .
Table 10-7
Interaction between serum cobalamin, serum folate, red cell folate, plasma homocysteine, serum methylmalonic acid, urinary methylmalonic acid and holotranscobalamin
| Clinical Status | Normal B 12 and Folate Status | B 12 Deficient | Folate Deficient |
|---|---|---|---|
| Serum B 12 * | Usually normal, but may be high in liver disease, myeloproliferative neoplasms, acute inflammation, recovery from autoimmune neutropenia. High levels may be due to immunoglobulin-complexed B 12 . Low total B 12 in 25% of elderly subjects. | Usually low, but up to 5% of patients with megaloblastic anaemia may have results within reference range | Usually normal, but low B 12 may be seen in severe folate deficiency, which corrects when monotherapy with folic acid is given |
| Serum folate * | Usually normal | Usually normal; high serum folate may occur in B 12 deficiency | Usually low, but normal levels are found with recent dietary improvement |
| Red cell folate * , † | Usually normal | Low | Usually low, but normal in very acute deficiency state |
| Plasma homocysteine | Usually normal; but may be high in renal failure or in MTHFR C677T mutation. Levels may fall with folate supplements. | High in B 12 deficiency and in 50% of samples from patients with low B 12 consistent with metabolic B 12 -deficient state | High in folate deficiency – corrected with folic acid therapy |
| Serum methylmalonic acid | Usually normal; high in 10% of normals and those with high intake of methionine or renal failure | High in B 12 deficiency and in 50% of samples from patients with low B 12 consistent with metabolic B 12 -deficient state | Usually normal, but high in 5% of patients who are folate deficient |
| Urinary methylmalonic acid | Normal even in renal failure | High | Normal |
| Holotranscobalamin | Normal although lower levels in elderly | Low (< 25 pmol/l) in B 12 deficiency | Normal |
* For normal reference values, see Chapter 2 .
† Folate assays may exhibit different responses to folic acid compared with methylenetetrahydrofolate. Assays may not be optimised for haemolysate matrix. Definition of reference range requires reference method and population studies.
Standards, accuracy and precision of cobalamin and folate assays
There are no internationally recognised reference methods for serum cobalamin measurement, but isotope dilution liquid chromatography tandem mass spectrometric methods are accepted as international reference methods for the quantification of folate species in serum. , As a result, international reference materials have been developed (by the World Health Organisation, WHO 03/178, and by the National Institute of Standards and Technology, NIST SRM). Although reference methods have not been verified for the whole blood matrix as yet, there is a WHO whole blood international standard (95/528) with consensus values for total folate.
Evaluation of commercial automated binding assays by recovery experiments has shown under-recovery of added 5-methyl THF and over-recovery of pteroylglutamic acid (PGA), whereas a suitably calibrated microbiological assay recovers closer to 100%. Differential sensitivity of assays to PGA and genetic variability in the proportion of in vivo formyl folates may be a factor in intermethod variability.
A microbiological assay was the method used to assign a potency value to the British Standard for human serum B 12 , and this was later reclassified as the 1st WHO International Standard (IS) (81/563). The 2nd WHO IS for serum B 12 , 03/178, was ratified in 2007, the values adopted being a consensus of the contemporary B 12 protein-binding assays. A consensus HoloTC value of 107 pmol/l for 03/178 was subsequently assigned using the Axis-Shield/Abbott Architect assay and was ratified in 2015.
External quality assessment schemes have shown serum vitamin B 12 intramethod coefficients of variation (CV) of 6–10% and as much as 20% at clinically relevant levels; there is thus a substantial ‘grey’ indeterminate range between normal and low values. Serum folate intramethod CVs are between 6% and 12% and higher CVs of up to 20% are seen for red cell folate assays. Overall between-method CVs may be as high as 35% for the serum methods and can reach 50% for the whole blood assays, suggesting considerable method differences.
Genetic factors
A number of polymorphisms in MTHFR , encoding methylenetetrahydrofolate reductase, that alter the proportion of formylfolate in serum have been described. Individuals homozygous for the MTHFR C677T polymorphism have 25% higher plasma homocysteine levels than controls. Cigarette smoking, age, renal disease, drugs including levodopa, and folate supplements all affect homocysteine levels.
Pre-analytical sample preparation
Serum vitamin B 12 is stable at room temperature, unless the sample is haemolysed. HoloTC is a sensitive marker of recent cobalamin intake and day-to-day variation is ≈ 10%. Folate is affected by recent dietary intake, and ideally fasting samples should be taken. Marked loss of folate activity is observed as a result of light and temperature instability. Because red cells contain 30–50 times more folic acid than serum, even slight haemolysis will affect serum folate analysis. Thus avoidance of haemolysis, rapid transportation and separation prior to analysis, avoidance of storage at room temperature and the storage of samples at 2–8 °C for a maximum of 48 h, or at − 20 °C for no longer than 28 days are all critical factors in the accuracy and precision of serum folate assays. The presence of haemoglobin as a result of lysis in a plasma or serum sample can be readily determined and may be quantified by haemoglobinometry.
The addition of sodium ascorbate 5 mg/ml will stabilise folate in serum, extending sample storage times, but necessitates introduction of separate B 12 and folate sample tubes since ascorbate interferes with cobalamin analysis. Samples must be fibrin free and without bubbles.
Analytical factors
Analytical sensitivity or limit of detection (LOD) varies between methods. It is defined as the concentration of analyte at 2SD of 20 replicates above the zero standard and for vitamin B 12 assays is normally in the region of 22 pmol/l (30 ng/l) and for folate in the region of 0.68 nmol/l (0.3 μg/l). This is sometimes confused with the functional sensitivity of an assay, a term that defines the analyte concentration at which the CV of the assay is 20%. It is preferable that the functional sensitivity limit of serum vitamin B 12 assays is closer to 37 pmol/l (50 ng/l) than the 111 pmol/l (150 ng/l) quoted by some kits because this provides increased sensitivity at the clinically important lower end of the reference range.
For many folate assays, functional sensitivity is in the region of 2.26 nmo1/l (1.0 μg/l) or less, although the Roche Elecsys assay quotes 4.5 nmol/l (2.0 μg/l).
Limitations and interference
Methotrexate and folinic acid interfere with folate measurement because these drugs cross-react with folate-binding proteins. Minor degrees of haemolysis significantly increase serum folate values as a result of high red cell folate levels. Lipaemia with > 2.25 mmol/l (2 g/l) of triglycerides and bilirubin > 340 μmol/l (200 mg/l) may affect assays.
‘Unusually’ high cobalamin levels are often due to vitamin B 12 therapy, vitamin supplementation, myeloproliferative neoplasms or liver disease. In the absence of these factors, assay interference may result from immunoglobulin–B 12 – transcobalamin complexes.
Post-analytical factors
The clinical interpretation of laboratory data should take account of the positive or negative predictive value of a result. The measurement of uncertainty should also be known and available to requesting users on request. The report should include a reference range, the derivation of which should also be readily available to users.
Methods for cobalamin and folate analysis
Microbiological bioassays and radiodilution assays for serum vitamin B 12 and folate are still used, albeit by a decreasing minority of laboratories, and continue to play an important role in the evaluation of new automated methods. They are also used in population studies where they are useful in providing information on the long-term comparability of results. (They are detailed in the 9th edition of this book.)
Modern methods are highly automated, heterogeneous, competitive protein-binding assays with chemiluminescence or fluorescence detection systems.
General principles of competitive protein-binding assays
The majority of automated single-platform, multianalyte, random-access analysers offer assays for serum vitamin B 12 , folate and homocysteine by nonisotopic competitive protein-binding or immunoassay. Second antibodies may be utilised as part of the separation procedures.
Serum B 12 assays
Release from endogenous binders and conversion of analyte to appropriate form
Approximately 99% of serum vitamin B 12 is bound to endogenous binding proteins (haptocorrin and transcobalamin) and must be released from these before measurement. The release step utilises alkaline hydrolysis (NaOH at pH 12–13) in the presence of potassium cyanide (KCN), which converts cobalamin to the more stable cyanocobalamin, and dithiothreitol (DTT) to prevent rebinding of released vitamin B 12 . Alkaline hydrolysis requires subsequent adjustment of pH to be optimal for the binding agent.
Binding of B 12 to kit binder
The binding of vitamin B 12 to kit binder is the competitive step of the assay. Serum-derived cyanocobalamin competes with labelled cobalamin, which is usually complexed to a chemiluminescent or fluorescent substrate or enzyme, for limited binding sites on porcine IF. Specificity for cobalamin is ensured by purification of the IF or by blocking contaminating corrinoid binders (R binders) by addition of excess blocking cobinamide. Specificity of pure and blocked IF can be demonstrated by the addition of cobinamide to sera. There should be no increase in assay value. Some assays use only the alkaline denaturation step to inactivate the endogenous binders. It is important that assays are not affected by the presence of high-titre anti-intrinsic factor antibody in patient sera. Examples of diagnostic failures with assays based on competitive-binding luminescence assays are widespread and have been associated with all the commonly used platforms. Some assays now carry product literature warnings to this effect. A study illustrated failure rates of cobalamin assays in the analysis of samples from patients with proven pernicious anaemia as a function of diagnostic platform: 6 of 23 (26%) Beckman Coulter Access assay, which used the UniCel DxI 800 Immunoassay System, 5 of 23 (22%) Roche Elecsys Systems Modular Analytics E170 and 8 of 23 (35%) Siemens Advia Centaur assay. The HoloTC assay by Axis-Shield is unaffected. , ,
Separation of bound and unbound B 12
Following competitive binding, the separation of bound and unbound vitamin B 12 is achieved by a number of electro- or physico-chemical and immunological methods. The Roche Elecsys utilises an electrochemiluminescence measuring cell in which the bound B 12 –ruthenium–IF complex, attached to paramagnetic particles by biotin–streptavidin, is magnetically captured onto the surface of an electrode. The Abbott Architect uses polymer microparticles (beads) with an iron core, coated with porcine IF to bind vitamin B 12 . The bound vitamin B 12 is then immobilised by positioning the assay reaction vessel in front of a magnet that pulls the paramagnetic microparticles onto the side wall of the reaction vessel. The reaction vessel contents are then aspirated and the reaction vessel refilled with buffer (in total the aspiration and refill step is repeated three times). These steps are designed to improve separation of bound and unbound B 12 .
Signal generation
The bound fraction is then detected by the addition of a chemiluminescent, fluorescent or colorimetric enzyme substrate, which results in generation of fluorescence or light emission. There are two types of signal: flash, which is pH or electrically induced, and plateau, which is sustained. The initial rate of reaction or the area under the curve is used to calculate the result.
You're Reading a Preview
Become a Clinical Tree membership for Full access and enjoy Unlimited articles
If you are a member. Log in here