BLOOD

Anaemia:

Anemia ( meaning lack of blood) is a decrease in number of red blood cells (RBCs) or less than the normal quantity of hemoglobin in the blood.

However, it can include decreased oxygen-binding ability of each hemoglobin molecule due to deformity or lack in numerical development as in some other types of hemoglobin deficiency.

The normal level of hemoglobin is generally

different in males and females. For men, anemia is

typically defined as hemoglobin level of less than

13.5 gram/100ml and in women as hemoglobin of

less than 12.0 gram/100ml.

These definitions may vary slightly depending on

the source and the laboratory reference used

What causes anemia• Any process that can disrupt the normal life span

of a red blood cell may cause anemia. Normal life span of a red blood cell is typically around 120 days. Red blood cells are made in the bone marrow.

• Anemia is caused essentially through two basic pathways. Anemia is either caused:

by a decrease in production of red blood cell or hemoglobin, or

by a loss or destruction of blood.

WHO's Hemoglobin thresholds used to define anemia (1 g/dL = 0.6206 mmol/L)

Age or gender group

Hb threshold (g/dl)Hb threshold

(mmol/l)

Children (0.5–5.0 yrs)

11.0 6.8

Children (5–12 yrs) 11.5 7.1

Teens (12–15 yrs) 12.0 7.4

Women, non-pregnant (>15yrs)

12.0 7.4

Women, pregnant 11.0 6.8

Men (>15yrs) 13.0 8.1

Peripheral blood smear microscopy of a patient with iron-deficiency anemia.

Classification:1. Microcytic :

Microcytic anemia is primarily a result of hemoglobin synthesis failure/insufficiency, which could be caused by several etiologies:

• Heme synthesis defect

– Iron deficiency anemia

– Anemia of chronic disease (more commonly presenting as normocytic anemia)

• Globin synthesis defect

– alpha-, and beta-thalassemia

– HbE syndrome

– HbC syndrome

– and various other unstable hemoglobin diseases

• Sideroblastic defect

– Hereditary sideroblastic anemia

– Acquired sideroblastic anemia, including lead toxicity

– Reversible sideroblastic anemia

2. Macrocytic :

• Megaloblastic anemia, the most common cause of macrocytic anemia, is due to a deficiency of either vitamin B12, folic acid (or both). Deficiency in folate and/or vitamin B12 can be due either to inadequate intake or insufficient absorption. Folate deficiency normally does not produce neurological symptoms, while B12 deficiency does.

Pernicious anemia is caused by a lack of intrinsic factor. Intrinsic factor is required to absorb vitamin B12 from food. A lack of intrinsic factor may arise from an autoimmune condition targeting the parietal cells (atrophic gastritis) that produce intrinsic factor or against intrinsic factor itself. These lead to poor absorption of vitamin B12.

Macrocytic anemia can also be caused by removal of the functional portion of the stomach, such as during gastric bypass surgery, leading to reduced vitamin B12/folate absorption.

Therefore one must always be aware of anemia following this procedure.

HypothyroidismAlcoholism commonly causes a macrocytosis, although not specifically anemia. Other types of Liver Disease can also cause macrocytosis.Methotrexate, zidovudine, and other drugs that inhibit DNA replication.Macrocytic anemia can be further divided into "megaloblastic anemia" or "non-megaloblastic macrocytic anemia". The cause of megaloblastic anemia is primarily a failure of DNA synthesis with preserved RNA synthesis, which result in restricted cell division of the progenitor cells. The megaloblastic anemias often present with neutrophil hypersegmentation (6–10 lobes). The non-megaloblastic macrocytic anemias have different etiologies (i.e. there is unimpaired DNA globin synthesis,) which occur, for example in alcoholism.

Normocytic:

Normocytic anemia:

Normocytic anemia occurs when the overall hemoglobin levels are always decreased, but the red blood cell size (Mean corpuscular volume) remains normal. Causes include:Acute blood lossAnemia of chronic diseaseAplastic anemia (bone marrow failure)Hemolytic anemia

Dimorphic:

When two causes of anemia act simultaneously, e.g., macrocytic hypochromic, due to hookworm infestation leading to deficiency of both iron and vitamin B12 or folic acid or following a blood transfusion more than one abnormality of red cell indices may be seen. Evidence for multiple causes appears with an elevated RBC distribution width (RDW), which suggests a wider-than-normal range of red cell sizes.

Heinz body anemia:

Heinz bodies form in the cytoplasm of RBCs and appear like small dark dots under the microscope.

There are many causes of Heinz body anemia, and some forms can be drug induced. It is triggered in cats by eating onionsor acetaminophen  (paracetamol). It can be triggered in dogs by ingesting onions or zinc, and in horses by ingesting dry red maple leaves

Refractory anemia :

Refractory anemia is an anemia which does not respond to treatment.  It is often seen secondary to myelodysplastic syndromes.Iron deficiency anemia may also be refractory as a clinical manifestation of gastrointestinal problems which disrupt iron metabolism

Iron regulation and metabolism

• Body iron content – 3-4g– Hb, iron containing proteins, bound to Tf,

storage (ferritin, haemosiderin).

• Iron homeostasis is regulated strictly at level of intestinal absorption.

Regulation of iron balance

• Haem diet – very readily absorbed via haem carrier protein 1 (apical bruish border membrane of duodenal enterocytes) i.e. higher bioavailability.

• Remainder of dietary iron poorly absorbed (10%).– Ascorbic acid enhances absorption of non-animal

sources of iron; tannates inhibit absorption.

• Fe2+ better absorbed cf. Fe3+.

Intestinal iron absorption

• Fe3+ freed from food binding sites in stomach, binds to mucin, travels to duodenum and small bowel.– Haem iron - carrier protein (endocytosis).– Fe3+ - attachment to an integrin.– Fe2+ - intestinal transporter DMT1.

Intestinal iron absorption

• Iron then enters cytosol, binds to cytosolic low molecular weight iron carriers and proteins e.g. Mobilferrin (shuttles iron with help of ATP) to basolateral membrane

• Export from basolateral membrane via duodenal iron exporter.

Intestinal iron absorption

• Upon release into circulation, re-oxidised to Fe3+, loaded onto transferrin.– Site of influence of HFE gene product, +/-

caeruloplasmin (known ferroxidase).

Iron transport

• Iron absorption regulated by many stimuli –– Iron stores.– Degree of erythropoiesis (increased with

increased erythropoiesis, reticulocytosis).– Ineffective erythropoiesis.– Mobilferrin – mechanism of loss in iron replete

state.

Iron regulation

Iron Absorption Procedure

Iron absorption in the human body takes place through the digestive tract, most importantly the small intestine. The remaining organs of the digestive system always contribute their bit to breaking down of food matter. However, the important part comes when the iron from the food source gets assimilated into the blood flow and into the hemoglobin. This process is aptly termed as the 'absorption' of iron. In human body, the small intestine is classified into different parts. The duodenum is the first part and is the place of iron absorption. This process of absorption is initiated by a class of cells that is termed as enterocytes. These cells are present in the inner glycocalyx surface of the duodenum, of the small intestine. The glycocalyx, is an extracellular polymeric surface that is secreted by the cells themselves. 

Moving on to the chemical procedure of the iron absorption, it must be noted that irons that are a part of the protein, are usually absorbed by the body. This 'absorbable' iron containing protein is often referred to as the heme protein. The ferrous form of this protein is chemically represented as Fe2+. Not all dietary irons are in this form, and some of them have to be reduced down from Fe3+. This function is conducted at the 'brush border', where a ferric reductase enzyme (a type of enzyme), duodenal cytochrome B, reduce it down to Fe2+. The process of conversion finishes here and a protein by the name DMT1, which is also known as divalent metal transporter 1, transports the iron into the cell. There are some very complex procedures that are involved in later stages where the iron optimizes the oxygen carrying capacity of the hemoglobin. 

IRON ABSORPTION Favored by

• Dietary factors: Increased Haem iron Increased animal iron Ferrous iron salts• Luminal factors: Acid pH (e.g. gastric HCl) Low molecular weight soluble

chelates (e.g. Vit. C, sugars, amino acids)• Ligand in meat (unidentified)• Systemic factors: Iron deficiency Increased erythropoiesis Ineffective erythropoiesis Pregnancy Hypoxia

Reduced by

Decreased haem ironDecreased animal ironFerric iron salts

Alkalis (e.g. pancreatic secretions)

Insoluble iron complexes (e.g. phytates, tannates in tea, bran)

Iron overloadDecreased erythropoiesisInflamatory disorders

• Transferrin and TfR.

• Ferritin.

• Iron responsive element-binding protein (IRE-BP) aka iron regulatory protein/factor (IRP/IRF).

• HFE.

• Divalent metal transporter (DMT1, Nramp2, DCT1,Slc11a) – duodenal iron transporter.

• Ferroportin and hephaestin, iron export proteins.

• Hepcidin.

Role of specific proteins

• Encoded on long arm of chromosome 3.

• Half life 8 days.

• Hepatic synthesis.

• Complete lack incompatible with life (hypotransferrinaemia).

Transferrin (Tf)

• Also on long arm of chromosome 3. homodimeric transmembrane protein.– Found in most cells. Most dense on erythroid

precursors, hepatocytes, placental cells.– Restricted expression: both TfR1 and TfR2

present at high levels in hepatocytes, epithelial cells of small intestine including duodenal crypt cells.

Transferrin receptor (TfR)

• Each TfR binds 2 diferric Tf molecules. Uptake by clustering on clathrin coated pits, then endocytosed.

• Iron off-loaded in acidified vacuoles, apotransferrin-TfR complex recycled to cell surface, apo-Tf then released back into circulation.

Transferrin receptor (TfR)

• Cellular storage protein for iron.

• L and H chains (chromosome 19, 11).

• Synthesis controlled at 2 levels – – DNA transcription via its promotor.– mRNA translation via interactions with iron

regulatory proteins.

• Acute phase reactant.

Ferritin

Ferritin in erythroid precursors may be of special importance in haem synthesis especially at beginning of Hb accumulation, when Tf-TfR pathway still in sufficient.

When ferritin accumulates, it aggregates, proteolyzed by lysosomal enzymes, , then converted to iron-rich, poorly characterised haemosiderin, which releases iron slowly.

M-ferritin – present in mitochondria. Expression correlated with tissues that have high mitochondrial number, rather than those involved in iron storage.

Ferritin

• Sensing iron-regulatory proteins modulate synthesis of TfR, ferritin, DMT1.– IRP1 and IRP2 – cytosolic RNA binding

proteins. Bind to iron-responsive elements located in 5’ or 3’ untranslated regions of specific mRNAs encoding ferritin, TfR, DMT1 and (in erythroid cells) eALAS.

Iron-regulatory proteins and iron-responsive element binding protein

Expression in GIT limited to cells in deep crypts in proximity to site of iron absorption.

HFE protein associated with TfR, acts to modulate uptake of Tf-bound iron into crypt cells.

Along with hepcidin, acts as iron sensor.Hereditary haemochromatosis with HFE gene

mutation - inability to bind beta 2-microglobulin, impaired cellular trafficking, reduced incorporation into the cell membrane, reduced association with TfR1.

HFE protein

• Divalent metal transporter protein – iron transporter (also Pb, Zn, Cu).

• Widely expressed, esp. in proximal duodenum.

• Isoform containing iron responsive element (Nramp2 isoform I) specifically upregulated in iron deficiency, greatest expression at brush border of apical pole of enterocytes in apical 2/3 of villi.

Duodenal iron transporter

• Increased body iron stores – enhanced uptake of iron from circulation into crypt cells.

• Increasing intracellular iron into crypt cells, differentiating enterocytes migrating up to villus tip downregulate iron transporter DMT1, reducing absorption of dietary iron from gut.

• Inverse relationship between ferritin levels in serum, and DMT1 levels in duodenal cells.

Duodenal iron transporter

• Transporting iron from basolateral membrane of enterocytes to circulation; from macrophage (from effete RBCs) into circulation for formation of new Hb.– Ferroportin.– Hephaestin.

Iron exporters

• Ferroportin-1 in basal portion of placental syncytiotrophoblasts, basolateral surface of duodenal enterocytes, macrophages, hepatocytes.

• Upregulated by amount of available iron, downregulated through interaction with hepcidin.

Ferroportin

• Homology to caeruloplasmin.

• Link between iron deficiency and copper deficiency – administration of copper facilitates progress of iron from tissue(s) into circulation.

Hephaestin

• SFT-mediated transport has properties defined for Tf-independent iron uptake, transporting iron across lipid bilayer. Process dependent on Cu.

• Has ferrireductase activity.

• Cytosolic localisation in recycling endosomes, stimulates Tf bound iron assimilation.

Stimulator of iron transport (SFT)

• 25 aa peptide hormone.

• Chromosome 19.

• Synthesized by hepatocyes. Intrinsic antimicrobial activity.

Hepcidin

• Binds ferroportin, complex internalised and degraded.

• Resultant decrease in efflux of iron from cells to plasma

Hepcidin

• Iron – stimulated with increased iron levels

• Inflammation, infection (and endotoxin)

• Hypoxia - downregulated

• Erythropoiesis – downregulated in anaemia, oxidative stress, ineffective erythropoiesis.

Regulation of hepcidin

• BMP - members of TGF-b superfamily which regulate cell proliferation, differentiation, apoptosis.

• Targets BMP receptors type I and II, resulting in phosphorylation of cytoplasmic R-Smads.

• R-Smads associate with Smad4, translocate to nucleus, activates transcription of target genes (in this case hepcidin).

Hepcidin regulation by bone morphogenic protein pathway

• Production stimulated by increased plasma iron and tissue stores.

• Negative feedback - hepcidin decreases release of iron into plasma (from macrophages and enterocytes).

• Fe-Tf increases hepcidin mRNA production (dose dependent relationship).

Hepcidin regulation by iron

TREATMENT

Iron : Iron is normally taken in the form of ferrous sulfate. Although other iron salts are commercially available and make claims of fewer or less severe side effects, these benefits may be related to the fact that other preparations contain less iron by weight. Ferrous sulfate contains about 37% iron, while ferrous gluconate contains only about 13% iron. People who have trouble with the side effects of ferrous sulfate may benefit from some of the specialty preparations available, but ferrous sulfate normally offers the greatest amount of iron of all commercial products.

Recommended dosage :

Dosage should be calculated by iron needs, based on laboratory tests.

Recommended one tablet a day,

containing 65 mg of iron, as a supplement for patients over the age of 12 years.

PrecautionsIron can lead to lethal poisoning in children. All iron supplements should be kept carefully out of reach of children.

Some types of anemia do not respond to iron therapy, and the use of iron should be avoided in these cases.

People with acquired hemolytic anemia, autoimmune hemolytic anemia, hemochromatosis, hemolytic anemia and hemosiderosis should not take iron supplements.

Iron supplements should also be avoided by people who have gastric or intestinal ulcers, ulcerative colitis, or Crohn's disease.

These conditions marked by inflammation of the digestive tract, which would be made worse by use of iron.

Side effects :

The most common side effects of iron consumption are stomach and intestinal problems, including stomach upset with cramps, constipation, diarrhea, nausea, and vomiting.

At least 25% of patients have one or more of these side effects. The frequency and severity of the side effects increases with the dose of iron. Less frequent side effects include heartburn and urine discoloration.

InteractionsIron supplements should not be taken at the same time as antibiotics of either the tetracycline or quinolone types. The iron will reduce the effectiveness of the antibiotic.

Also, iron supplements reduces the effectiveness of levodopa, which is used in treatment of Parkinson's disease.

Iron supplements should not be used with magnesium trisilicate, an antacid, or with penicillamine, which is used for some types of arthritis.

Taking iron with vitamin C increases the absorption of iron, with no increase in side effects.

Folic acid :

Folic acid is found in many common foods, including liver, dried peas, lentils, oranges, whole-wheat products, asparagus, beets, broccoli, brussel sprouts, and spinach.

However, in some cases, patients have difficulty absorbing folic acid or in converting it from the form found in foods to the form that is active in blood formation.

In these cases, folic acid tablets are appropriate for use.

Recommended Dose : For treatment of anemia, a daily dose of 5 mg is generally used. Patients who have trouble absorbing folic acid may require higher doses.

Maintenance doses are:

infants: 0.1 mg/day

children (under 4 years of age): up to 0.3 mg/day

children (over 4 years of age) and adults: 0.4 mg/day

pregnant and lactating women: 0.8 mg/day

Precautions:

Before treating an anemia with folic acid, diagnostic tests must be performed to verify the cause of the anemia. 

Pernicious anemia caused by lack of vitamin B12 shows symptoms that are very similar to those of folic acid deficiency but also causes nerve damage which shows up as a tingling sensation and feelings of numbness.

Giving folic acid to patients with B12 deficiency anemia improves the blood cell count, but the nerve damage continues to progress.

SIDE EFFECTS.

Folic acid is considered extremely safe, and there are no predictable side effects.

Where side effects have been reported, they have been among patients taking many times more than the normal therapeutic dose of the drug.

On rare occasions allergic reactions to folic acid have been reported.

INTERACTIONS.

Phenytoins, used to treat seizure disorders, interact with folic acid with a reduction in phenytoin effectiveness and an increased risk of seizures.

If the two drugs must be used together, phenytoin blood levels should be monitored, and the dose may have to be increased.

Trimethoprim (an antibacterial) and Methotrexate (originally an anti-cancer drug, which is also used for arthritis and psoriasis) act by reducing the metabolism of folic acid.

Regular blood monitoring is required, and dose adjustments may be needed.

Vitamin B12 is also known as cyanocobalamine and hydroxocobalamine.

Cyanocobalamine may be given by mouth, while hydroxocobalamine must be injected.

The vitamin has many functions in the body, including maintaining the nervous system, but in treatment of anemia B12 is needed for the metabolism of folic acid. Lack of B12 causes pernicious anemia, a type of anemia which is marked by a low red cell count and lack of hemoglobin.

There are many other symptoms of pernicious anemia, including a feeling of tingling or numbness,shortness of breath, muscle weakness, faintness, and a smooth tongue. If pernicious anemia is left untreated for more than three months, permanent damage to the nerves of the spinal cord may result.

Recommended Dose :

While vitamin B12 can be given by mouth for mild vitamin deficiency states, pernicious anemia should always be treated with injections, either under the skin (subcutaneous) or into muscle (intramuscular). Hydroxocobalamine should only be injected into muscle. Intravenous injections are not used because the vitamin is eliminated from the body too quickly when given this way.

Elderly patients, whose ability to absorb vitamin B12 through the stomach may be impaired, should also be treated with injections only.The normal dose of cyanocobalamine is 100 mcg (micrograms) daily for six to seven days. If improvement is seen, the dose may be reduced to 100 mcg every other day for seven doses and then 100 mcg every three to four days for two to three weeks. After that, monthly injections may be required for life.

PRECAUTIONS.

Although vitamin B12 has a very high level of safety, commercial preparations may contain preservatives which may cause allergic responses.

In patients with pernicious anemia, treatment with vitamin

B12 may lead to loss of potassium. Patients should be monitored for their potassium levels.

SIDE EFFECTS.

Diarrhea and itching of the skin have been reported on rare occasions.

Moreover, there have been reports of severe allergic reactions to cyanocobalamine.

INTERACTIONS.

Aminosalicylic acid may reduce the effectiveness of vitamin B12.

Also, Colchicine, a drug used for gout, may reduce the effectiveness of vitamin B12.

Other, infrequently used drugs and excessive use of alcohol may also affect the efficacy of vitamin B12.

Anabolic steroids :The anabolic steroids (nandrolone, oxymetholone, oxandrolone, and stanzolol) are the same drugs that are used improperly by body builders to increase muscle mass.

Two of these drugs, nandrolone and oxymetholone, are approved for use in treatment of anemia.

Nandrolone is indicated for treatment of anemia caused by kidney failure, while Oxymetholone may be used to treat anemia caused by insufficient red cell production, such as aplastic anemia.

All anabolic steroids are considered to be drugs of abuse under F.D.A and only recommended under certain circumstances.

RECOMMENDED DOSAGE.

The information that follows is specific only to oxymetholone; however, the warnings and precautions apply to all drugs in the class of anabolic steroids.

The dosage of oxymetholone must be individualized.

The most common dose is 1 to 2 mg per kilogram of body weight per day, although doses as high as 5 mg per kilogram per day have been used.

The response to these drugs is slow, and it may take several months to see if there is any benefit.

PRECAUTIONS.

All anabolic steroids are dangerous. The following warnings represent the most significant hazards of these drugs.

Peliosis hepatitis, a condition in which liver and sometimes spleen tissue is replaced with blood-filled cysts, has occurred in patients receiving androgenic anabolic steroids.

Although this condition is usually reversible by discontinuing the drug, if it is left undetected and untreated, it may lead to life-threatening liver failure or bleeding.

Liver tumors may develop. Although most of these tumors are benign and will go away when the drug is discontinued, liver cancers may also result.

Anabolic steroids may cause changes in blood lipids, leading to atherosclerosis with greatly increased risk of heart attack.

Because anabolic steroids are derived from male sex hormones, masculinization may occur when they are used by women.

Elderly men who use these drugs may be at increased risk of prostate enlargement and prostate cancer.

Increased water retention due to anabolic steroids may lead to heart failure.

Anabolic steroids should not be used during pregnancy, since this may cause masculinization of the fetus.

Anabolic steroids should be used in children only if there is no possible alternative. These drugs may cause the long bones of the legs to stop growing prematurely, leading to reduction in adult height. Regular monitoring is essential.

In patients with epilepsy, the frequency of seizures may be increased.

In patients with diabetes, glucose tolerance may be altered. Careful monitoring is essential.

SIDE EFFECTS.

The list of side effects associated with anabolic steroids is extremely long. The following list covers only the most commonly observed effects:

acneincreased urinary frequencybreast growth in malesbreast painpersistent, painful erectionsmasculinization in women

INTERACTIONS :

Anabolic steroids should not be used in combination with anticoagulants such as warfarin.

Anabolic steroids increase the effects of the anticoagulant, possibly leading to bleeding.

If the combination cannot be avoided, careful monitoring is essential.

Erytropoetin :

Erythropoietin is a glycoprotein hormone that controls erythropoiesis, or red blood cell production. It is a cytokine for erythrocyte (red blood cell) precursors in the bone marrow and has its primary effect on red blood cells by promoting red blood cell survival through protecting these cells from apoptosis. It also cooperates with various growth factors involved in the development of precursor red cells. And A similar drug, darepoetin alpha, is available with the same properties, but it remains active longer and so requires fewer injections each week.

Epoetin alpha is approved by the Food and Drug Administration for the following uses:

anemia associated with chronic renal failureanemia related to zidovudine therapy in HIV-infected patientsanemia in cancer patients on chemotherapyreduction in blood transfusions in surgical patients

RECOMMENDED DOSAGE.:

Dosing schedules may vary with the cause of the anemia.

All doses should be individualized.

In general, epoetin alpha dosing in adults is started at 50 to 100 units per kilogram given three times a week, either by vein or subcutaneously.

PRECAUTIONS.

Epoetin alpha should not be given to patients with severe, uncontrolled hypertension.

Other conditions in which epoetin alpha should be used only when the benefits clearly outweigh the risks are as follows:

constitutional aplastic anemiahypertensionthromboembolism

Side effects :

The most common adverse effects of erythopoetin alpha are:joint painchest paindiarrheaswellingfatiguefeverweaknessheadachehigh blood pressureirritation at injection sitenauseavomitingrapid heart beat

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