Anemias

Classification of Anemia

Pathophysiological

· Decreased production

o

No Reticulocyte response

Marrow infiltration, injury

o Nutritional deficiency

o Erytropoeitn deficiency

o Ineffective

· Acute blood loss

·

Reticulocyte response

Hemolysis

o Extrinsic to RBC acquired

§ Intravascular

§ Extravascular

o Intrinsic to RBC inherited

Erythrocyte size

· Microcytic

· Normocytic

· Macrocytic

Marrow response to Anemia

	Polychromasia The cell indicated by the arrow does not contain inclusions but is bluer than the rest of the cells. Ribonucleic acid, which is found in young red cells, gives it the blue color. The bluish cells are reported as polychromasia. Polychromasia (a mixture of colors-the eosinophilia of hemoglobin and the bluishness of ribonucleic acid) is correlated with the number of reticulocytes. Reticulocytes, however, cannot be seen in Wright's stained smears and must be stained with a vital stain before they become visible. If much polychromasia is present, the observer knows that there is an increased number of reticulocytes.
	Reticulocyte A reticulocyte is any non-nucleated cell of the erythrocytic series containing RNA, which when stained with new methylene blue will have discernible granules or have a diffuse network of fibrils. They are the hallmark of erythrocyte regenerative response. Polychromasia: Polychromatophylic erythrocytes are those that show a faint bluish tint due to an admixture of the characteristic colors of hemoglobin and the basophilic erythrocyte cytoplasm when stained with a Romanowsky stain or a quick stain. Many of these would be reticulocytes if stained with new methylene blue. Polychromasia is an indicator of regenerative anemia. Reticulocyte: A reticulocyte is any non-nucleated cell of the erythrocytic series containing RNA, which when stained with new methylene blue will have discernible granules or have a diffuse network of fibrils. They are the hallmark of erythrocyte regenerative response.
	Nucleated Erythrocytes Nucleated red blood cells are commonly observed in regenerative anemias, but may also be observed in non-anemic or non-regenerative anemic states such as lead poisoning, hypoxia, or myeloproliferative disease.
	Howell-Jolly Bodies: HJ bodies usually single, are nuclear (DNA) remnants observed in young erythrocytes. They may be observed in splenic disease or in the absence of a spleen, since the spleen normally removes HJ bodies from red cells
	Basophilic Stippling Basophilic stippling is observed in erythrocytes stained with Wright-Giemsa, Romanowsky stains such as the quick stains, seen in a diverse group of red cell disorders, and small numbers are seen on normal peripheral blood smears. Pathobiology: The stippled material is composed of RNA and represents aggregates of ribosomes. Differential diagnosis: Thalassemias (stippling may be coarse) Megaloblastic anemias Lead and other heavy metal poisoning (stippling is coarse) Dyserythropoiesis of whatever etiology (stippling usually fine) Unstable hemoglobinopathies Liver disease (stippling fine) Hereditary pyrimidine 5'-nucleotidase deficiency (stippling coarse)

RBC Count   x 10⁶/mcL
Male      3.93 - 5.69
Female  3.67 - 5.06

Reticulocyte Count % (SI:  fraction= 0.01 x %)

0.66 - 2.47
Retic Absolute K/mcL:       31.7 - 104.6

Reticulocyte Index = reticulocyte (%) x (patient’s Hct/Normal Hct) normal response >3%

DMT-1

Production of the transferrin receptor (TfR), DMT-1 and ferritin is regulated at the level of mRNA by iron regulatory proteins (IRPs), which bind to iron response elements (IREs) on the 3'- and 5'- untranslated regions of their respective mRNAs. A) In iron deficiency, the IRPs bind to the IREs, protecting the TfR mRNA from nuclease digestion and preventing the synthesis of ferritin. B) When iron is abundant, the modified IRP no longer binds to the IREs — in IRP1 the IRE binding site is blocked by a 4Fe–4S cluster (green rectangle), whereas in IRP2 the protein is targeted for destruction in the proteasome — allowing TfR mRNA to be destroyed and allowing the expression of ferritin.

Absorption of Iron by Enterocyte.

Hepcidin

Figure 1. Hepcidin as a regulator of iron trafficking. Blocks release of Iron from intenstinal mucosa and macrophages

Hepcidin as a regulator of iron trafficking.(A) In normal subjects, circulatory iron sets a basal level of hepcidin synthesis by hepatocytes. Serum hepcidin modulates the amount of iron released from macrophages and enterocytes that contributes the pool of circulatory iron able, in a regulatory feedback loop, to control the hepatic production of hepcidin. HFE, the product of the hemochromatosis gene, is probably required for hepcidin activation in response to the circulatory iron signal. Other hemochromatosis gene products (i.e. HJV and TfR2) might also be involved.

(B) During iron deficiency and anemia (hypoxia), hepcidin transcription is turned off and circulating levels of the peptide drop: enhanced release-transfer of iron from storage sites and the intestine follows. It is presently unclear whether HFE (and other hemochromatosis proteins) are required for downregulation of hepcidin during iron deficiency

(C) During inflammatory states interleukin-6 (IL-6) released from macrophages (including Kupffer cells) induces hepcidin transcription and, as a consequence, high circulatory levels of the peptide: iron is sequestered in macrophages and intestinal iron transfer is decreased. This leads to circulatory hypoferremia and, in some cases, to anemia (anemia of inflammation or chronic disease). Seemingly, during circulatory iron overload, hepcidin synthesis, in the presence of functional HFE (and TfR2 and HJV), is upregulated and the release of iron from storage sites and the intestine halted.

(D) As HFE is required for upregulation of hepcidin transcription in response to circulatory iron, if HFE is nonfunctional (i.e. HFE-related hereditary hemochromatosis) hepcidin synthesis by the hepatocytes is unregulated and inappropriately low: the consequent unrestricted release of iron from macrophages and enterocytes leads to progressive expansion of the plasma iron pool followed by tissue iron overload and organ damage. Circumstantial evidence indicates that HJV (and possibly TfR2) might also be required for iron sensing by hepatocytes. Therefore, a similar pathogenic pathway may be shared by HJV- and TfR2-related hemochromatosis.

Two main pathways of iron acquisition in animal cells.
(a) Uptake of Tf-bound iron in reticulocytes and other cells expressing TfR1 involves Steap3 and DMT1. (b) Uptake of non–Tf-bound iron in intestine and other cells mediated by Dcytb and DMT1. Asc, ascorbate; DHA, dehydroascorbic acid; e, electron.

Causes of Microcytic anemia

Common:

Iron Deficiency

Thalassemia

Less common:

Anemia of inflammation (normally normocytic)

Hemoglobin C

Hemoglobin E

Hereditary Pyropoikilocytosis

Lead normally normocytic

Rare

Sideroblastic anemia

Copper deficiency

Congenital atransferrinemia

Causes of reduced serum iron:

Fe def

Infection

Connective tissue disease

Cancer

Post op stress

Stress

Normal

Red cell distribution

~16.3

Fe doses

Neonates require 1mg/kg/day

3mg/kg/day elemental iron for mild

5-6mg/kg/day elemental iron for severe

Ferrous sulphate 20% elemental iron (Fe2+)

1 week retics up, Hb up 1gm/dl

4-6 weeks to completely correct

Total 3-4 months total therapy needed.

Prevention of Iron Deficiency

Premature infants need extra

Breast feed for first 6 months

Avoid cows milk first 12 months

Iron fortified formula 5-10% bioavailability

Breast 50% bioavailability

Limit cows milk after 18-24oz (500-672mls)/day

28% breast fed babies are Fe def at 9 months

Iron Content of Food

Megaloblastic Anemia

	Normal blood is shown on the right and the blood from a patient with pernicious anemia on the left. Notice the large ovalocytes typical of megaloblastic anemia. Macrocytic cells usually are seen in patients with 13,2 or folic acid deficiency, but can be seen in other conditions such as myeloid metaplasia, refractory megaloblastic anemia, liver disease, hypothyroidism, and after treatment with some antimetabolites.
	Vitamin B12 Cobalamin Deficiency causes neurological symptoms, loss of joint position sense, ataxia, psychomotor retardation seizures, seizures, psychosis.

BM of megaloblastic anemia

	hypercellularity of megaloblastic anemia
	Decreased myeloid/erythroid ratio of megaloblastic anemia Granulocyte precursors of megaloblastic anemia many being larger than normal, including giant bands and metamyelocytes.
	RBC precursors of megaloblastic anemia abnormally large and have nuclei that appear much less mature than would be expected from the development of the cytoplasm (nuclear-cytoplasmic asynchrony).
	Megaloblastic anemia. Folate deficiency. Blood film. There is no morphologic distinction in the blood or marrow appearance of cells in megaloblastic anemia as a result of vitamin B12 or folate deficiency. Oval macrocytes, anisocytosis, and poikilocytosis are characteristic of each etiology of megaloblastic anemia. Note also hypersegmented neutrophil

Macrocytosis Causes

Normal newborn

Reticulocytosis

Marrow failure

Drugs AZT, methotrexate

Cyanotic congenital heart disease

Downs syndrome

Hypothyroidism

Liver Disease

Megaloblastic anemias (B12,folate)

Laboratory Diagnosis of Folate and Cobalamin deficiency

Serum folate (current levels)

RBC folate (tissue levels)

Serum Vitamin B12

Schilling test

Stage 1

Administer 0.5-2.0 mCi of radioactive cyanocobalamin in a glass of water to fasting patients.

Two hours later, the patient is injected with 1 mg of unlabeled vitamin B-12 to saturate circulating transcobalamins.

A 24-hour urine sample is collected, and the radioactivity in the specimen is measured and compared to a standard.

Specimens with less than 7% excretion represent abnormal findings and indicate that poor absorption of the oral test dose occurred.

Stage II Schilling test

If abnormal low values are obtained, a stage II Schilling test is performed. In this test, 60 mg of active hog IF is administered with the oral test dose to determine if this enhances the absorption of vitamin B-12. If poor absorption of vitamin B-12 is normalized, the patient presumably has classic pernicious anemia.

If poor absorption is observed in a stage II test, search for other causes of vitamin B-12 malabsorption. Performance of a stage I Schilling test after 5 days of tetracycline therapy is used to exclude a blind loop as the etiology for Cbl deficiency (stage III). Similarly, if administration of trypsin or pancreatic enzyme with the radiolabeled test dose corrects the absorption of vitamin B-12, suspect pancreatic disease (stage IV).

False-positive Schilling test results are observed in patients with incomplete 24-hour urine collections or renal insufficiency, false-positive results are observed when inactive IF is used, and false-positive results occur because of neutralization of the IF in the stage II test by any IF antibodies in the stomach and severe ileal megaloblastosis.

Occasionally Cbl deficiency and a normal result on stage I Schilling test are observed. These patients can absorb vitamin B-12 in the fasting state but not when it is presented with food.

Adding the radiolabeled vitamin B-12 to egg white and testing the absorption usually reveals this cause of Cbl deficiency.

Other causes of Megaloblastic Anemia

Hereditary Orotic aciduria

Congenital Dyserythropoietic Anemia

Melodysplasia

M6 AML

Congenital Dyserythropoietic Anemia as a cause of macrocytosis

	Congenital Dyserythropoietic Anemia HEMPAS Nuclear bridging, 100x
	Congenital Dyserythropoietic Anemia HEMPAS Multinuclearity, 100x

Methemoglobinemia:

Erythrocytes (RBCs) possess 4 hemoglobin chains, each of which contain a heme moiety. These hemoglobin chains function to transport and deliver oxygen to tissues. Methemoglobin can be found in RBCs when there is oxidation (ie, loss of an electron) of the iron moiety, changing the normal oxygen-carrying ferrous (Fe²⁺) state to the ferric (Fe³⁺) state. Ferric heme is incapable of binding oxygen because of a stoichiometric alteration of the molecule. However the O2 affinity of the rest of the Hemes are increased (O2 disassociation curve shifted to left).

Pathophysiology:

• Oxidation of iron to the ferric state reduces the oxygen-carrying capacity of hemoglobin and produces a functional anemia.
• In addition, methemoglobinemia shifts the hemoglobin dissociation curve to the left.
• Ferric heme groups impair the release of oxygen from ferrous heme groups on the same hemoglobin tetramer; thus, oxygen delivery to tissues is impaired.
• Methemoblobin is reduced in the blood by NADH catalyzed by cytochrome B₅ reductase (=methemoglobin reductase, NADH diaphorase). NADH made by Emden-Meyerhof pathway.
• Drugs and chemicals like alanine dyes and nitrates from well contamination
• Infant of diabetic mothers and IUGR patients prone to this.
• Young children
• 10-40% Cyanosis
• 50-70% Cardiovascular collapse and death
• Treatment mehytlene blue (do not use with GAPD deficiency, causes massive hemolysis)- boards question!
• Treatment includes ascorbic acid.

Lab Studies:

• Bedside test: To distinguish between deoxyhemoglobin and metHb, place 1 or 2 drops of the patient's blood on a white filter paper. Deoxyhemoglobin brightens after exposure to atmospheric oxygen, but metHb does not change color. Blowing oxygen on the filter paper speeds the reaction.

• The limitation of arterial blood gas (ABG) is that metHb can falsely elevate the calculated oxygen saturation.

• Pulse oximetry: Findings on bedside pulse oximetry are misleading. This device only measures the relative absorbance of 2 wavelengths of light to differentiate oxyhemoglobin from deoxyhemoglobin; however, metHb absorbs both of these wavelengths equally. Therefore, at high levels of metHb, the pulse oximeter reads a saturation of 85%, which corresponds to equal absorbance of both wavelengths. This is an inaccurate depiction of the Hb oxygen-carrying capacity. Also important to note is that the partial pressure of oxygen (pO₂) value on the ABG reflects plasma oxygen content, does not correspond to the oxygen-carrying capacity of Hb, and should be within the reference range in patients with methemoglobinemia.

• Co-oximetry: The co-oximeter is an accurate method for measuring metHb and is the key to diagnosing metHb. It is a simplified spectrophotomer that can measure the relative absorbance of 4 different wavelengths of light and, therefore, can differentiate metHb from carboxyhemoglobin, oxyhemoglobin, and deoxyhemoglobin. Newer machines also can measure sulfhemoglobin. One problem is that not all clinical care laboratories have these machines.

• Potassium cyanide test: This test can distinguish between metHb and sulfhemoglobin. MetHb reacts with cyanide to form cyanometHb, which has a bright red color. Sulfhemoglobin does not react with cyanide and therefore does not change to a bright red color.

• Tests to rule out hemolysis (eg, CBC, reticulocyte counts, lactate dehydrogenase [LDH], indirect bilirubin, haptoglobin) and to test for organ failure and general end-organ dysfunction (eg, liver function tests, electrolytes, BUN, creatinine)

• Tests to evaluate a hereditary cause for metHb should be ordered when appropriate, eg, Hb electrophoreses and NADH-dependent metHb reductase levels.

Hemoglobin M

A group of abnormal hemoglobins in which amino acid substitutions take place in either the alpha or beta chains but near the heme iron. This results in facilitated oxidation of the hemoglobin to yield excess methemoglobin which leads to cyanosis.

Methemoglobin is an oxidized form of hemoglobin, which is unable to carry oxygen and lead to cyanosis.
Methemoglobinemia is a situation in which the level of methemoglobin exceeds 1%. Normally, about 3% of the total hemoglobin is daily oxidized, but already reduced by the enzymatic cytochrome b5 reductase system.
Two types of methemoglobinemia need to be distinguished:

 in the first one, which may be a severe disease, normal hemoglobin is oxidized into methemoglobin. This may result from an increased formation of methemoglobin through the action of a toxic agent, to a deficiency in cytochrome b5 reductase activity (recessive congenital methemoglobinemia -RCM- of type I or II), or to the presence of a hemoglobin variant with increased auto-oxidation rate.

 The second type of methemoglobinemia is due to the presence of a variant with abnormal spectral properties named Hb M. Hbs M, were the first hemoglobin variants described. Seven structural abnormalities leading to Hb M have been described as shown in the table below. They concern the distal (E7) or proximal (F8) histidines of the , or chain, which is replaced by a tyrosine. In Hb M Milwaukee [ 67 (E11) Val -> Glu], the residue modified is located near to the distal histidine.
These variants are found worldwide, and result frequently from de-novo mutations.

Name	Mutation	Clinical presentation
Hb M-Boston	58 (E7) His->Tyr ( 1 or 2)	cyanosis starts at birth and remains at a constant level. Well tolerated
Hb M-Iwate	87 (F8) His->Tyr ( 1 or 2)	cyanosis starts at birth and remains at a constant level. Well tolerated
Hb M-Saskatoon	63 (E7) His ->Tyr	cyanosis develops after birth and reach its final level at 6 months. Well tolerated
Hb M-Hyde Park	92 (F8) His->Tyr	cyanosis develops after birth and reach its final level at 6 months. This Hb is also unstable and leads to some degree of hemolytic anemia
Hb M-Milwaukee	67 (E11) Val -> Glu	cyanosis develops after birth and reach its final level at 6 months. Well tolerated
Hb F-M-Osaka	G 63 (E7) His->Tyr	Cyanosis is maximum at birth and deceases progressively with the switch from HbF to Hb A
Hb F-M-Fort Ripley	G 92 (F8) His->Tyr	Cyanosis is maximum at birth and deceases progressively with the switch from HbF to Hb A

Hb M should be considered in all patients with chronic cyanosis, especially when their pulmonary and cardiac function is normal. The greatest hazard for carriers of Hb M is misdiagnosis with the risk of expensive and hazardous cardiovascular investigations and this is specially the case for newborn babies.

Laboratory diagnosis

The presence of Hb M is suggested by a typical chocolate brownish color of the blood. Hematological parameters are normal except for Hb M-Hyde Park. Analysis of the hemolysate by isoelectric-focusing reveals an abnormal brownish band migrating close to the position of Hb F.
RP-HPLC is a good tool to confirm the presence of a HbM variant since the mutation His->Tyr lead to a clear increase in hydrophobicity.

A more specific diagnosis is made through spectrophotometrical studies. As compared to normal methemoglobin the absorption peaks in the visible are shifted towards a shorter wavelength (610-620 nm instead of 630 nm for normal methemoglobin). This abnormal spectrum may be difficult to recognize in the case of an or chain variant, since it only affects the abnormally oxidized hemes - ca. 10% of the total heme here.

Sulfhemoglobinemia

Sulfhemoglobinemia is a rare condition in which there is excess sulfhemoglobin (SulfHb) in the blood. The pigment is a greenish derivative of hemoglobin which cannot be converted back to normal, functional hemoglobin. It causes cyanosis even at low blood levels.

Sulfhemoglobinemia is usually drug induced. Drugs associated with sulfhemoglobinemia include acetanilid, phenacetin, nitrates, trinitrotoluene and sulfur compounds (mainly sulphonamides). Another possible cause is occupational exposure to sulfur compounds. The condition generally resolves itself with erythrocyte (red blood cell) turnover, although blood transfusions can be necessary in extreme cases.