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Path physiology hematology

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Merck Manual red blood cell indices MCV MCH MCHC RDW iron deficiency anemia hepcidin ferroportin

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Hematology: RBC Indices and Iron Deficiency Anemia

1. Red Blood Cell (RBC) Indices

RBC indices are calculated from:

Hemoglobin (Hb)
Hematocrit / packed cell volume (Hct/PCV)
RBC count

They help classify anemia morphologically.

A. Mean Corpuscular Volume (MCV)

Definition: Average volume of an individual RBC.

Normal range: 80-100 fL

Formula:

[ \text{MCV (fL)} = \frac{\text{PCV/Hct (%)} \times 10}{\text{RBC count (million/µL)}} ]

MCV	Type of anemia	Common causes
<80 fL	Microcytic anemia	Iron-deficiency anemia, thalassemia, sideroblastic anemia, anemia of chronic inflammation/disease
80-100 fL	Normocytic anemia	Acute blood loss, hemolytic anemia, aplastic anemia, early anemia of chronic disease
>100 fL	Macrocytic anemia	Vitamin B12 deficiency, folate deficiency, liver disease, alcohol use, hypothyroidism

Key point: MCV is the principal RBC index used to classify anemia by cell size.

B. Mean Corpuscular Hemoglobin (MCH)

Definition: Average amount, or mass, of hemoglobin in one RBC.

Normal range: 27-33 pg

Formula:

[ \text{MCH (pg)} = \frac{\text{Hb (g/dL)} \times 10}{\text{RBC count (million/µL)}} ]

C. Mean Corpuscular Hemoglobin Concentration (MCHC)

Definition: Average concentration of hemoglobin in a given volume of packed RBCs.

Normal range: 32-36 g/dL

Formula:

[ \text{MCHC (g/dL)} = \frac{\text{Hb (g/dL)} \times 100}{\text{PCV/Hct (%)}} ]

A low MCHC indicates hypochromia, commonly seen in iron-deficiency anemia.

D. Red Cell Distribution Width (RDW)

Definition: A measure of variation in RBC size.

Increased variation in RBC size is called anisocytosis.
Normal RDW: approximately 11.5-14.5%

A raised RDW is common in iron deficiency because both older normal-sized cells and newly produced smaller cells may circulate together.

2. Packed Cell Volume (PCV) / Hematocrit (Hct)

Definition: Percentage of total blood volume occupied by RBCs.

Method:

[ \text{PCV} = \frac{\text{Height of packed RBC column}}{\text{Total height of blood column}} \times 100 ]

Approximate normal ranges:

Male: 40-54%
Female: 36-46%

Relationship between Hct, RBC count, and MCV:

[ \text{Hct (%)} \approx \frac{\text{RBC count (million/µL)} \times \text{MCV (fL)}}{10} ]

3. Common Reference Values

Test	Typical adult reference range
Hemoglobin, male	13-17 g/dL
Hemoglobin, female	12-15 g/dL
RBC count, male	4.5-6.0 million/µL
RBC count, female	4.0-5.5 million/µL
WBC count	4,000-11,000/µL
Platelet count	150,000-450,000/µL, or 1.5-4.5 lakh/µL

Ranges differ slightly between laboratories and with age, altitude, and pregnancy.

4. Anemia

Definition: Anemia is a reduction in the oxygen-carrying capacity of blood, usually identified by a hemoglobin concentration below the appropriate reference range for age, sex, and physiological state.

Anemia is not defined simply as “deficiency of Hb and RBC count.” Hb is the primary diagnostic measure. RBC count can be low, normal, or even high, such as in thalassemia trait.

5. Iron-Deficiency Anemia

Iron-deficiency anemia is typically a microcytic, hypochromic anemia.

Causes / Etiology

Inadequate dietary iron intake
Increased requirement
- Pregnancy
- Infancy, childhood, and adolescence
Chronic blood loss
- Heavy menstrual bleeding
- Gastrointestinal blood loss, such as peptic ulcer disease, malignancy, or hemorrhoids
Parasitic infestation
- Especially hookworm in endemic areas
Malabsorption
- Celiac disease
- Reduced gastric acid or post-gastrectomy states
- Other small-intestinal disorders
Reduced intake or impaired absorption associated with alcohol use, usually in the context of poor nutrition or gastrointestinal/liver disease

In adults, especially adult men and postmenopausal women, iron deficiency should prompt evaluation for a source of chronic blood loss, commonly from the gastrointestinal tract. The Merck review of iron-deficiency anemia provides the typical laboratory pattern and causes.

6. Intestinal Iron Absorption and Regulation

Iron absorption pathway

Dietary non-heme iron is mainly present as ferric iron (Fe³⁺).
At the brush border of the duodenum, duodenal cytochrome b (Dcytb) reduces Fe³⁺ to ferrous iron (Fe²⁺).
DMT1 transports Fe²⁺ into the enterocyte.
Iron can be stored within enterocytes as ferritin or exported into blood through ferroportin.
During export, Fe²⁺ is oxidized to Fe³⁺, chiefly by hephaestin.
Fe³⁺ binds transferrin for transport in plasma to tissues, especially bone marrow, liver, and other cells.

Hepcidin-ferroportin regulation

Hepcidin is made by the liver.
It binds to ferroportin and causes ferroportin internalization and degradation.
Therefore, increased hepcidin reduces:
- Intestinal iron absorption
- Release of iron from macrophages
- Release of stored iron from hepatocytes

Important correction to the handwritten note:

High hepcidin does not cause iron deficiency directly.
In uncomplicated iron deficiency, hepcidin decreases. This allows ferroportin to remain active and increases iron absorption and release into plasma.
Hepcidin is increased by high iron stores and inflammation, contributing to iron-restricted erythropoiesis in anemia of chronic inflammation.

7. Signs and Symptoms of Anemia

Common features include:

Fatigue
Weakness
Reduced exercise tolerance
Dizziness or light-headedness
Pallor
Dyspnea on exertion
Palpitations or tachycardia in more severe anemia

Features that can suggest iron deficiency include:

Pica, especially craving ice
Brittle nails or koilonychia
Glossitis
Angular cheilitis
Hair loss in some people

Epigastric pain is not a direct usual symptom of anemia, but may point to an underlying cause of chronic blood loss, such as peptic ulcer disease.

8. Blood Film Terminology

Anisocytosis: Variation in RBC size
Poikilocytosis: Variation in RBC shape

Quick Summary

MCV measures cell size: low in microcytic anemia, normal in normocytic anemia, and high in macrocytic anemia.
MCH measures hemoglobin amount per RBC.
MCHC measures hemoglobin concentration within packed RBCs.
RDW measures RBC size variation and rises with anisocytosis.
Iron-deficiency anemia is usually microcytic and hypochromic, often with increased RDW.
Major causes of iron deficiency are poor intake, increased physiological demand, chronic blood loss, hookworm, and malabsorption.
Iron is absorbed mainly in the duodenum as Fe²⁺ through DMT1, exported by ferroportin, and transported by transferrin.
Hepcidin inhibits ferroportin. Hepcidin is generally low in pure iron deficiency and high during inflammation.

Megaloblastic anemia absorption cycle normal and deficiency both detailed with picture

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diagram of vitamin B12 absorption stomach intrinsic factor terminal ileum cubilin receptor

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megaloblastic anemia peripheral blood smear macro ovalocytes hypersegmented neutrophils image

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vitamin B12 deficiency AND megaloblastic anemia

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Merck Manual megaloblastic macrocytic anemia vitamin B12 folate absorption deficiency

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An accurate labeled medical teaching diagram of vitamin B12 and folate absorption and the folate-methionine DNA synthesis cycle. Show B12 path: animal food, stomach acid and pepsin release B12, haptocorrin, pancreatic enzymes in duodenum, intrinsic factor from parietal cells, terminal ileum cubilin receptor uptake, transcobalamin II transport to cells and liver stores. Show folate path: vegetables and fortified foods, jejunum absorption. Then a cellular inset: B12-dependent methionine synthase converts homocysteine to methionine and converts 5-methyl THF to THF; folate provides 5,10-methylene THF for dUMP to dTMP DNA synthesis. Include deficiency blocks for pernicious anemia or ileal disease and folate malnutrition/malabsorption, leading to impaired DNA synthesis, nuclear-cytoplasmic asynchrony, megaloblastic anemia, macro-ovalocytes, hypersegmented neutrophils. Include a clear note that B12 deficiency also causes high methylmalonic acid and neurologic disease, while folate deficiency does not. Use correct terminology, simple textbook style, no patient-specific details.

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Megaloblastic Anemia: Vitamin B12 and Folate

Vitamin B12 and folate absorption, metabolic cycle, and deficiency

Definition

Megaloblastic anemia is a macrocytic anemia caused by defective DNA synthesis, most commonly due to deficiency of:

Vitamin B12 (cobalamin)
Folate (vitamin B9)

DNA synthesis is impaired, so the nucleus matures and divides slowly while cytoplasmic maturation continues normally. This is called nuclear-cytoplasmic asynchrony. It affects rapidly dividing cells, particularly bone marrow precursors and gastrointestinal epithelial cells.

Typical blood-film findings are:

Macro-ovalocytes
Hypersegmented neutrophils, usually 5 or more lobes
Anisocytosis and poikilocytosis
Sometimes leukopenia and thrombocytopenia

Robbins & Kumar Basic Pathology, p. 396.

Hypersegmented neutrophil in megaloblastic anemia

1. Normal Vitamin B12 Absorption Cycle

Dietary source

Vitamin B12 is mainly present in animal-derived foods:

Meat
Fish
Eggs
Milk and dairy products

Body stores are large, especially in the liver. Thus, B12 deficiency generally takes years to appear after absorption fails.

Steps of absorption

Step 1: Stomach

Dietary B12 is bound to food proteins.
Gastric acid and pepsin release B12 from these proteins.
Free B12 binds to haptocorrin, also called R-binder or R-protein, which is produced in saliva and stomach.

Step 2: Duodenum

Pancreatic proteases digest haptocorrin.
B12 is released and binds to intrinsic factor (IF).
Intrinsic factor is a glycoprotein made by gastric parietal cells.

Step 3: Terminal ileum

The B12-IF complex reaches the terminal ileum.
It binds to the cubilin receptor complex on ileal enterocytes.
The complex is taken into enterocytes by receptor-mediated endocytosis.

Step 4: Blood and tissues

B12 is released from the enterocyte and enters plasma attached to transcobalamin II.
It travels to:
- Bone marrow for erythropoiesis
- Liver for storage
- Nervous tissue and other cells

Vitamin B12 requires intrinsic factor and is absorbed in the ileum. Costanzo Physiology, 7th ed., p. 388. B12 stores can delay anemia for 3 to 4 years after absorption is impaired. Guyton and Hall Textbook of Medical Physiology, p. 456.

2. Normal Folate Absorption Cycle

Dietary source

Folate is found in:

Green leafy vegetables
Legumes
Citrus fruits
Liver
Fortified grains

Food folate is often present as polyglutamate folate.

Absorption

Dietary polyglutamate folate is converted at the intestinal brush border into absorbable monoglutamate forms.
Folate is absorbed mainly in the duodenum and proximal jejunum.
In blood it is transported to tissues.
In cells, folate is converted to its active form, tetrahydrofolate (THF).

Unlike B12, body folate stores are limited. Deficiency can develop in weeks to months when intake, absorption, or utilization is inadequate. Folate is absorbed predominantly in the proximal small intestine. Yamada’s Textbook of Gastroenterology, 7th ed.

3. Folate-B12 Metabolic Cycle and DNA Synthesis

Both vitamins are essential for formation of thymidine (dTMP), required for DNA synthesis.

A. Folate cycle

[ \text{Folate} \rightarrow \text{DHF} \rightarrow \text{THF} \rightarrow 5,10\text{-methylene THF} ]

Dihydrofolate reductase (DHFR) converts dihydrofolate, DHF, to THF.
THF accepts and transfers one-carbon units.
5,10-methylene THF donates a methyl group in the reaction:

[ \text{dUMP} \rightarrow \text{dTMP} ]

dTMP is required to make DNA.

If folate is deficient, dTMP formation falls, DNA replication is impaired, and megaloblastic anemia develops.

B. Role of vitamin B12: the methyl-folate trap

B12 acts as a cofactor for methionine synthase:

[ \text{5-methyl THF} + \text{Homocysteine} \xrightarrow[\text{B12}]{\text{Methionine synthase}} \text{THF} + \text{Methionine} ]

Why B12 deficiency causes megaloblastic anemia

When B12 is deficient:

5-methyl THF cannot be converted back to THF.
Folate becomes trapped as 5-methyl THF.
The cell has less usable THF for DNA synthesis.
dTMP synthesis declines.
Nuclear maturation fails.
Megaloblasts develop in the bone marrow.

This is called the methyl-folate trap hypothesis.

4. B12's Additional Pathway

B12 is also needed for conversion of methylmalonyl-CoA to succinyl-CoA:

[ \text{Methylmalonyl-CoA} \xrightarrow[\text{B12}]{\text{Methylmalonyl-CoA mutase}} \text{Succinyl-CoA} ]

Therefore, B12 deficiency causes:

Increased methylmalonic acid (MMA)
Increased homocysteine

Folate deficiency causes:

Increased homocysteine
Normal MMA

This distinction is useful when laboratory results are uncertain.

5. Vitamin B12 Deficiency

Causes

Reduced intake

Strict vegan diet without B12 supplementation
Severe malnutrition

Defective gastric phase

Pernicious anemia: autoimmune destruction of parietal cells and/or antibodies against intrinsic factor
Atrophic gastritis
Total or partial gastrectomy
Gastric bypass surgery
Long-term acid suppression may contribute in susceptible people

Defective intestinal absorption

Terminal ileal disease, especially Crohn disease
Ileal resection
Bacterial overgrowth
Fish tapeworm infection
Pancreatic insufficiency, which impairs haptocorrin digestion

Clinical features

General anemia features

Fatigue
Weakness
Pallor
Dyspnea on exertion
Palpitations

Gastrointestinal features

Glossitis: sore, smooth, red tongue
Reduced appetite
Weight loss
Diarrhea

Neurologic features: unique to B12 deficiency

Peripheral neuropathy
Paresthesia
Loss of vibration and position sense
Ataxia
Cognitive or psychiatric changes
Subacute combined degeneration of the spinal cord in severe deficiency

Neurologic manifestations do not occur in uncomplicated folate deficiency.

Laboratory pattern

Investigation	B12 deficiency
MCV	High, often >100 fL
Peripheral smear	Macro-ovalocytes, hypersegmented neutrophils
Serum B12	Low
Homocysteine	High
Methylmalonic acid	High
LDH	High due to ineffective erythropoiesis
Indirect bilirubin	May be high
Reticulocyte count	Usually low
WBC and platelets	May be reduced

6. Folate Deficiency

Causes

Reduced dietary intake

Poor nutrition
Alcohol use disorder
Elderly or socially vulnerable patients
Prolonged overcooking of food

Increased requirement

Pregnancy
Lactation
Infancy and adolescence
Chronic hemolytic anemia
Malignancy

Malabsorption

Celiac disease
Proximal small-bowel disease
Extensive small-bowel surgery
Bariatric surgery

Drugs affecting folate

Methotrexate
Trimethoprim
Pyrimethamine
Phenytoin
Sulfasalazine

Features

Folate deficiency causes megaloblastic anemia that is hematologically similar to B12 deficiency:

Macro-ovalocytes
Hypersegmented neutrophils
Glossitis
Weakness and fatigue
Sometimes diarrhea

However, it does not cause B12-type neurologic disease.

Laboratory pattern

Investigation	Folate deficiency
MCV	High
Serum folate	Low
Homocysteine	High
Methylmalonic acid	Normal
Neurologic signs	Absent

7. B12 versus Folate Deficiency

Feature	Vitamin B12 deficiency	Folate deficiency
Main absorption site	Terminal ileum	Duodenum and proximal jejunum
Need for intrinsic factor	Yes	No
Body stores	Large, lasts years	Limited, depleted in months
Homocysteine	Increased	Increased
Methylmalonic acid	Increased	Normal
Neurologic manifestations	Present or possible	Absent
Common causes	Pernicious anemia, ileal disease/resection, vegan diet	Poor diet, alcohol, pregnancy, malabsorption, drugs
Blood film	Macro-ovalocytes, hypersegmented neutrophils	Same
Anemia treatment	B12 replacement	Folate replacement after ruling out B12 deficiency

8. Important Clinical Warning

Do not give folic acid alone until vitamin B12 deficiency has been excluded.

Folate can improve the anemia caused by B12 deficiency, but it does not correct the neurological injury. Neurologic damage may continue and can become irreversible. This warning is supported in the Merck guidance on megaloblastic anemia.

One-line exam summary

Vitamin B12 and folate deficiency impair thymidine synthesis and DNA replication, causing nuclear-cytoplasmic asynchrony, megaloblastic bone marrow, macro-ovalocytes, and hypersegmented neutrophils. B12 deficiency additionally raises methylmalonic acid and causes neurologic manifestations.

Permicius anemia

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Pernicious Anemia

Pernicious anemia is a megaloblastic anemia caused by vitamin B12 deficiency due to autoimmune loss of intrinsic factor.

It is the late hematologic manifestation of autoimmune metaplastic atrophic gastritis.

Basic mechanism

[ \text{Autoimmune destruction of parietal cells} \rightarrow \downarrow\text{Intrinsic factor} \rightarrow \downarrow\text{B12 absorption in terminal ileum} \rightarrow \text{B12 deficiency} \rightarrow \text{impaired DNA synthesis} \rightarrow \text{megaloblastic anemia} ]

Vitamin B12 absorption and deficiency pathway

Normal B12 absorption

Dietary vitamin B12 is released from food in the stomach.
It initially binds haptocorrin (R-protein).
In the duodenum, pancreatic enzymes degrade haptocorrin.
B12 binds intrinsic factor (IF), which is secreted by gastric parietal cells.
The B12-IF complex travels to the terminal ileum.
It binds ileal receptors and B12 is absorbed.
B12 is transported in blood by transcobalamin II and stored mainly in the liver.

Costanzo Physiology, 7th ed., p. 388.

What goes wrong in pernicious anemia?

Autoantibodies act against:

Gastric parietal cells, especially the H+/K+-ATPase
Intrinsic factor

This produces:

Atrophy of gastric body and fundus mucosa
Low gastric acid or achlorhydria
Reduced or absent intrinsic factor
Failure of B12 absorption in the terminal ileum

The ileum itself may be normal, but it cannot absorb B12 because the vitamin is not bound to intrinsic factor.

Causes and associations

Cause

Autoimmune metaplastic atrophic gastritis

Common autoimmune associations

Autoimmune thyroid disease, especially Hashimoto thyroiditis
Type 1 diabetes mellitus
Vitiligo
Addison disease

Autoimmune gastritis also increases the risk of gastric neuroendocrine tumors and gastric adenocarcinoma. The Merck review of autoimmune atrophic gastritis notes both B12 malabsorption and this long-term gastric cancer risk.

Clinical features

Symptoms of anemia

Fatigue and weakness
Pallor
Dyspnea on exertion
Palpitations
Dizziness

Gastrointestinal signs

Glossitis: smooth, red, painful tongue
Reduced appetite
Weight loss
Diarrhea may occur

Neurologic features of B12 deficiency

These distinguish B12 deficiency from folate deficiency:

Tingling or numbness in hands and feet
Loss of vibration and joint-position sense
Gait ataxia
Weakness
Memory, mood, or cognitive changes
Subacute combined degeneration of the spinal cord in advanced disease

Peripheral blood and bone marrow findings

CBC

Low hemoglobin
Increased MCV, often >100 fL
Reticulocyte count low
Leukopenia and thrombocytopenia may occur in severe disease

Peripheral smear

Macro-ovalocytes
Hypersegmented neutrophils with 5 or more nuclear lobes
Anisocytosis and poikilocytosis

Megaloblastic anemia arises from impaired DNA synthesis and may cause pancytopenia and raised LDH due to intramedullary destruction of erythroid precursors. Frameworks for Internal Medicine, p. 336.
Macro-ovalocytes and hypersegmented neutrophils are characteristic. Robbins & Kumar Basic Pathology, p. 396.

Laboratory findings

Test	Typical finding
Serum vitamin B12	Low
MCV	High
Serum methylmalonic acid	High
Homocysteine	High
LDH	High
Indirect bilirubin	May be high
Anti-intrinsic factor antibody	Positive, highly specific
Anti-parietal cell antibody	Sensitive but less specific
Serum gastrin	Often high due to achlorhydria
Pepsinogen I	Low in advanced gastric atrophy

Antibody interpretation

Anti-intrinsic factor antibody: More specific, but may be absent in some cases.
Anti-parietal cell antibody: More sensitive but not specific, since it may be positive in other autoimmune or gastric disorders.

Differential diagnosis

Disorder	B12	MMA	Homocysteine	Neurologic signs
Pernicious anemia / B12 deficiency	Low	High	High	May be present
Folate deficiency	Normal	Normal	High	Absent
Non-megaloblastic macrocytosis, such as alcohol or liver disease	Usually normal	Normal	Usually normal	Not due to B12 deficiency

Treatment

Treatment is vitamin B12 replacement for life, because the intrinsic-factor defect is usually permanent.

Common regimens use:

Intramuscular hydroxocobalamin or cyanocobalamin initially, then maintenance injections at regular intervals
High-dose oral B12 may be used in selected patients under medical supervision, because passive absorption can still occur

A commonly taught regimen is cyanocobalamin 1000 micrograms IM weekly for 4 weeks, then monthly, but local protocols differ and treatment should be prescribed based on the patient’s severity, neurological involvement, and local guidelines. Harrison’s Principles of Internal Medicine, 22nd ed.

Important caution

Do not treat a suspected B12 deficiency with folic acid alone.

Folate may correct the anemia but will not correct neurological injury, which can progress and may become irreversible. This is also emphasized in the Merck megaloblastic anemia guidance.

One-line exam answer

Pernicious anemia is an autoimmune atrophic gastritis causing loss of gastric parietal cells and intrinsic factor, leading to impaired terminal-ileal absorption of vitamin B12 and resulting in megaloblastic anemia with possible neurological manifestations.

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