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Haematinics Interpretation

Haematinics Interpretation

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Introduction

Haematinics are essential nutrients for producing blood cells in the process of haematopoiesis. Lack of them can be linked to cytopenia(s) and associated symptoms, while an abundance can imply an underlying disorder. This guide will discuss deficiencies in the most clinically relevant haematinics: vitamin B12 (cobalamin), vitamin B9 (folate) and iron.

Other haematinics not included in this guide are: vitamin A, vitamin B2 (riboflavin), vitamin B3 (nicotinic acid), vitamin B6, vitamin C, vitamin E, copper and cobalt.

It is imperative to take into account the patient's history and examination findings when interpreting a haematinics test. The relevance and implications of the results can vary depending on the context and should be analysed alongside a thorough clinical assessment.

Iron

Investigating a patient's iron status can be tricky as no single test accurately reflects all stages of iron metabolism. Additionally, some available tests are affected by inflammation and renal diseases.

The prevalence of iron deficiency anaemia makes it necessary to understand the tests used to detect it and interpret the results accordingly.

When to investigate

Iron deficient individuals can manifest microcytic anaemia and numerous symptoms such as fatigue, shortness of breath, weakness, reduced exercise tolerance, pica (e.g. ice craving) and restless leg syndrome.

During the physical examination, typical findings of iron-deficiency anaemia may include general and conjunctival pallor, atrophic glossitis, angular cheilitis and, less commonly, koilonychia (spoon-shaped nails).

Initial screening investigations

If iron deficiency anaemia is suspected, initial screening investigations should comprise a full blood count (FBC), C-reactive protein (CRP) and serum ferritin.

Full blood count (FBC) and red cell parameters

A full blood count allows the identification and quantification of anaemia through haemoglobin (Hb) and haematocrit (Hct) levels, although it does not provide insight into iron stores. To evaluate iron status, analyse the mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC) which may be low in iron deficiency, while red cell distribution width (RDW) may be elevated. These findings are non-specific and may not be present in every case.

Serum ferritin

Ferritin is an intracellular protein complex that binds iron and is responsible for most iron storage in the body. Small amounts of ferritin are secreted into the serum to transport and absorb any remaining iron, acting as a surrogate serum marker for body iron storage. There is a direct correlation between serum ferritin levels and overall iron stores in health.

Interpreting serum ferritin

Serum ferritin is an acute-phase protein. Its levels increase in inflammatory states, chronic kidney disease, liver disease and malignancy.

According to the British Society for Haematology (BSH) guidelines, a serum ferritin level of less than 15 μg/l is indicative of an iron deficiency in those aged 5 years and older. If the level is less than 150 μg/l and a patient has an concurrent inflammatory condition (acute or chronic) or renal impairment, further investigations should be considered.

The BSH suggests that C-reactive protein be part of the initial screening test, as a serum ferritin within the normal range may not exclude iron deficiency if a patient has raised inflammatory markers or a history of acute or chronic illness.

Secondary investigations

If the first screening tests are inconclusive but a suspicion of iron deficiency remains, the BSH recommends requesting secondary investigations, including transferrin saturations and a blood film.

Transferrin saturations

Transferrin saturation is the ratio of total serum iron (or the total iron-binding capacity) to transferrin expressed as a percentage. Transferrin is the primary serum iron transporter molecule in the body. In an iron-deficient state, more transferrin is produced to increase the total iron-binding capacity and acquire more iron for cells.

There is no hard evidence to support a diagnostic threshold, however the BSH consensus guidelines suggest a level of less than 16% as supportive of a diagnosis of iron deficiency if initial tests are inconclusive.

Blood film

A blood film can be useful if there is diagnostic uncertainty and can show morphological changes which may support the conclusion that a patient is iron deficient.

Blood Film Features of Iron Deficiency

Certain aspects of a blood film analysis can indicate that a patient is more likely to be suffering from iron deficiency. These features include anisocytosis, microcytosis, hypochromia, pencil cells, target cells, and elliptocytes.

Advanced Red Cell Parameters

Modern analysers can measure various advanced red cell parameters. These parameters may vary depending on the analyser but usually include mean reticulocyte haemoglobin concentration, the percentage of red cells that are hypochromic, and reticulocyte haemoglobin equivalent. British Society of Haematology suggests these parameters can be used to aid in secondary testing and a mean reticulocyte haemoglobin of <29 pg can be supportive of a diagnosis of iron deficiency, and may help predict iron response in patients with chronic kidney disease.

Other Investigations of Iron Metabolism

Though not necessary to diagnose iron-deficiency anaemia, a range of other investigations are available from laboratories and can be used to support the diagnosis.

Serum Iron

Serum iron measures only a fraction of the iron in the blood, and can only measure the ferric form (Fe3+), not iron incorporated in haemoglobin molecules. Serum iron levels show diurnal variation and are sensitive to recent iron intake, making it ineffective for determining a patient's iron stores.

Total Iron-Binding Capacity (TIBC)

The TIBC is calculated by taking a serum sample and adding excess iron to fully saturate the iron carrying molecules. This provides a measure of the total iron concentration in the sample when fully saturated. However, TIBC can rise in an iron-deficient state and as such has limited specificity and British Society of Haematology does not recommend its use as a routine tool for measuring iron stores.

Transferrin

Transferrin is the main serum iron transporter molecule that can be measured in a patient's serum. Also like TIBC, transferrin can rise in iron deficiency, though it is a negative acute-phase protein so it can be decreased in inflammatory states.

Soluble Transferrin Receptor (STFR)

Transferrin receptors, found on developing red cells, can be detected in soluble form in peripheral blood. In iron deficiency, these cells display more receptors to acquire more iron, which can be measured in the serum. Though STFR is not affected by inflammation, its lack of specificity, high cost, and inability to detect mild deficiency make it not recommended by British Society of Haematology for measuring iron stores.

Summary

The table below summarises how iron studies can be interpreted.

Table 1. Interpreting iron studies.

Iron Deficiency Anaemia and Vitamin B12 Deficiency

Iron deficiency anaemia

  • Ferritin: ↓/Normal
  • Transferrin/TIBC: ↑
  • Transferrin saturations: ↓
  • STFR: ↑/Normal

Treatment of Iron Deficiency Anaemia

For most patients, oral iron is a safe, effective and cost-effective method for treating iron deficiency. Co-administration of vitamin C improves the absorption of oral iron. Traditional dosing guidelines suggested aiming for 100-200mg of elemental iron a day, but more recent studies have shown that once-daily dosing of around 45-80mg elemental iron is more effective and reduces gastrointestinal side effects. This amount of elemental iron roughly equates to one tablet of either ferrous fumarate 210mg or ferrous sulphate 200mg. A response in the haemoglobin count should occur within a few weeks of starting therapy. In certain cases, intravenous iron may be preferred.

Vitamin B12

Testing for a patient's vitamin B12 and folate status is challenging as existing investigations are not sensitive or specific and show wide variability between different laboratories. For this reason, routine screening is not indicated for any patient group. Instead, investigations should be performed in response to specific clinical indicators. As vitamin B12 and folate share a close relationship in human metabolism and so present with similar features, they should be investigated simultaneously.

Clinical features of Vitamin B12 Deficiency

B12 deficiency often develops over years, with clinical features occurring insidiously. Patients with B12 deficiency can present with features related to anaemia such as fatigue, shortness of breath, weakness, and reduced exercise tolerance as well as neuropsychological features (motor and sensory peripheral neuropathies, ataxia, retinopathy, optic atrophy, cognitive impairment etc.) and glossitis. In severe deficiency, subacute degeneration of the spinal cord can develop. Importantly, not all patients with clinical vitamin B12 deficiency will have anaemia or macrocytosis. Therefore, if a patient presents with clinical features of B12 deficiency, is in an at-risk population or has been identified to have macrocytic anaemia, investigations of B12 stores should be undertaken. Infants can also present with faltering growth, movement disorders or developmental delay.

Causes of Vitamin B12 Deficiency

Causes of vitamin B12 deficiency include:

  • Malabsorption: pernicious anaemia, Crohn's, gastrectomy
  • Dietary deficiency: veganism, poor diet, alcohol misuse
  • Drug-induced: metformin, proton pump inhibitors

Vitamin B12 Deficiency

Vitamin B12 deficiency can occur due to a variety of causes, including certain medications, dietary deficiencies, infections and congenital causes. Specific scenarios with their associated causes of vitamin B12 deficiency are discussed below.

Specific scenarios

Medications

Certain medications can affect serum B12 assays creating misleading results. However, they rarely lead to clinically significant deficiencies, and B12 levels should only be checked in the presence of objective features of deficiency. Medications that decrease serum B12 levels include Metformin, anti-convulsants, proton pump inhibitors/H2 antagonists, hormone replacement therapy/combined oral contraceptive pill, colchicine and certain antibiotics.

Gastrointestinal Surgery

Patients who have undergone gastric surgery or bariatric surgery can often develop vitamin B12 deficiency. These patients should undergo monitoring of B12 levels and adopt a diet rich in B12 (red meat, salmon, milk, cheese etc.). Intramuscular replacement should be commenced if levels are falling despite this.

Pregnancy

A physiological reduction in serum B12 levels can occur in up to 30% of individuals by the third trimester of pregnancy, complicating assessment. If in doubt about the relevance of results, seek specialist advice.

Multiple myeloma

The presence of paraproteins can reduce serum B12, but rarely reflects clinical deficiency.

Primary testing

Serum cobalamin is the primary investigation suggested and should only be requested in the presence of objective clinical markers of deficiency. When investigating a patient for vitamin B12 deficiency, the initial blood tests should include serum cobalamin, full blood count, and a blood film.

Serum cobalamin

The most common investigation in the UK for vitamin B12 is serum cobalamin, but levels do not always correlate clinically. It is possible to be clinically deficient in vitamin B12 with a normal range serum cobalamin level and vice versa. False normal results can occur in the presence of very high titres of anti-intrinsic factor antibodies in pernicious anaemia. Falsely low serum levels may occur in concurrent folate deficiency or conditions listed above in the specific scenarios section. While there is no agreed normal level, most UK labs use a cut-off of <148pmol/l as evidence of B12 deficiency if objective clinical features are present.

Full blood count (FBC) and red cell parameters

Features which might suggest a vitamin B12 deficiency include anaemia and macrocytosis. However, it is possible to be B12 deficient with a normal full blood count. Reticulocyte counts will be low or normal as the bone marrow cannot make new cells without vitamin B12.

Blood film

A blood film can help establish the diagnosis and assess for concurrent diseases. Features on blood film are non-specific, but can include macrocytosis, hypersegmented neutrophils and oval macrocytes.

Investigating the cause

If a patient with B12 deficiency has a family history of pernicious anaemia or a personal history of an autoimmune condition, it is important to investigate for pernicious anaemia. BSH suggest screening for pernicious anaemia in patients with both clinical features and laboratory confirmed B12 deficiency.

Anti-intrinsic factor antibodies (Anti-IF)

Anti-intrinsic factor antibodies are found in pernicious anaemia and have a low false-positive rate with a high positive predictive value. They are present in 40-60% of pernicious anaemia cases. False-positive results can occur following recent B12 injections.

Anti-Gastric Parietal Cell Antibodies

Anti-gastric parietal cell antibodies are less specific, found in 10% of the general population who do not have pernicious anaemia, but up to 80% of those with the disease.

Secondary Testing

When the patient presents with convincing clinical features of vitamin B12 deficiency (e.g. macrocytic anaemia and glossitis or neurological symptoms) but has normal serum cobalamin levels, then secondary testing may be required.

Secondary testing for B12 deficiency includes:

  • Total plasma homocysteine: raised in B12 deficiency, but can also be elevated in folate deficiency, B6 deficiency, renal disease and hypothyroidism.
  • Plasma methylmalonic acid: raised in B12 deficiency and in renal disease, haemoconcentration and small bowel overgrowth.
  • Holotranscobalamin: more sensitive but not available in all labs. It is low in vitamin B12 deficiency.

Interpreting Secondary Tests for B12 Deficiency

The following table outlines the differences between B12 and Folate deficiencies:

  • Plasma Homocysteine: B12 Deficiency: ↑ Folate Deficiency: ↑
  • Plasma Methylmalonic Acid: B12 Deficiency: ↑ Folate Deficiency: –
  • Holotranscobalamin: B12 Deficiency: ↓ Folate Deficiency: –
Treatment of B12 Deficiency

In the United Kingdom, the treatment of B12 Deficiency consists of intramuscular (IM) Vitamin B12 injections. The dosage and frequency depend on the severity of symptoms and response:

  • If no neurological symptoms: 1mg IM three doses a week for 2 weeks followed by every 3 months
  • If neurological symptoms present: 1mg IM alternative days until no further improvement in symptoms (minimum 3 weeks) followed by every 2 months

Once treatment begins, a rise in the reticulocyte count should be seen within the first 7-10 days.

Folate

Folate is the collective term for all biological forms of Vitamin B9, while Folic Acid is the synthetic form used to treat deficiencies. Both forms are absorbed in the terminal ileum and half of the body's folate is stored in the liver. Folate is often tested at the same time as Vitamin B12 as they are closely related in human metabolism and have similar deficiency symptoms.

Folate is essential for DNA synthesis, making rapidly dividing cells of the body the first to be affected by deficiency. Symptoms of folate deficiency can include fatigue, weakness, mouth ulcers, and glossitis which are all related to anaemia.

Isolated folate deficiency is rare, however if the patient tests positively for both Vitamin B12 and folate deficiencies, B12 treatment should be administered first as any other action can result in subacute degeneration of the spinal cord.

Causes of Folate Deficiency

Folate deficiency can be caused by conditions that inhibit absorption, increase secretion, or increase the body's folate requirements:

  • Dietary: Deficient in folate-rich foods
  • Alcoholism: Alcohol intake > 80g/10 units a day
  • Gastrointestinal Disorders: Coeliac or any disorder affecting the small bowel
  • Pregnancy: Preferential delivery of folate to foetus
  • Haematological Disorders: Sickle Cell Anaemia or abnormal haematopoiesis
  • Exfoliative skin disorders: Psoriasis
  • Medications: Anti-epileptics

Primary Testing

When folate deficiency is suspected, the primary tests include serum folate levels and full blood count.

Serum Folate

Serum folate is the most useful initial screening test. Deficiency is generally defined as a level <7nmol/L, which is associated with an increased risk of megaloblastic anaemia. Levels of 7-10nmol/L suggest a grey area, in which case a treatment trial may be helpful.

It is important to note that serum folate can produce false results in anorexia, acute alcohol intake, pregnancy, anti-epileptic therapy, and post-haemodialysis.

Folate Deficiency

Falsely high levels of folate can occur if the test is performed too soon after oral folate ingestion.

Full Blood Count/Blood Film

The lack of folate affects developing red cells and leads to the presence of large, abnormal red cells called megaloblasts in peripheral blood. This causes an increased MCV on the full blood count, which is a late consequence.

Secondary Testing

If the clinical suspicion of folate deficiency is high but serum folate is in the normal range, BSH guidelines suggest considering secondary testing. These can include:

  • Red cell folate: lots of laboratory variables limiting use, time-consuming and expensive
  • Plasma total homocysteine: high in folate deficiency but non-specific as can be raised in B12 deficiency, B6 deficiency, renal disease and hypothyroidism

Treatment of Folate Deficiency

If a patient is folate deficient, it is important to assess for concurrent vitamin B12 deficiency.

If a patient is deficient in both folate and B12, they should be started on B12 replacement first to avoid precipitating or worsening neurological features of B12 deficiency. The usual treatment of folate deficiency is folic acid 5mg once daily for 3-4 months unless the cause of the folate deficiency is chronic (e.g. haemolysis) in which case a longer duration of treatment might be required.

References

  • Fletcher, A., Forbes, A., Svenson, N., Wayne Thomas, D. (2022), Guideline for the laboratory diagnosis of iron deficiency in adults (excluding pregnancy) and children. Br J Haematol, 196: 523-529.
  • Devalia, V., Hamilton, M.S., Molloy, A.M. and (2014), Guidelines for the diagnosis and treatment of cobalamin and folate disorders. Br J Haematol, 166: 496-513.
  • Camaschella C. (2015) Iron-deficiency anemia. New England journal of medicine. May 7;372(19):1832-43.

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