Collection of blood:

Venous blood is most commonly used for a majority of biochemical investigations. It can be drawn from any prominent vein (usually from a vein on the front of the elbow).

Capillary blood (<0.2 ml) obtained from a finger or thumb, is less frequently employed.

Arterial blood (usually drawn under local anesthesia) is used for blood gas determinations.

Precautions for blood collection : Use of sterile (preferably disposable) needles and syringes, cleaning of patients skin, blood collection in clean and dry vials/tubes are some of the important precautions.

Biochemical investigations can be performed on 4 types of blood specimens – whole blood, plasma, serum and red blood cells. The selection of the specimen depends on the parameter to be estimated.

1. Whole blood (usually mixed with an anticoagulant) is used for the estimation of hemoglobin, carboxyhemoglobin, pH, glucose, urea, non-protein nitrogen, pyruvate, lactate, ammonia etc. (Note : for glucose determination, plasma is prefered in recent years).

2. Plasma, obtained by centrifuging the whole blood collected with an anticoagulant, is employed for the parameters—fibrinogen, glucose, bicarbonate, chloride, ascorbic acid etc.

3. Serum is the supernatant fluid that can be collected after centrifuging the clotted blood. It is the most frequently used specimen in the clinical biochemistry laboratory. The parameters estimated in serum include proteins (albumin/globulins), creatinine, bilirubin, cholesterol, uric acid, electroylets (Na+, K+, Cl-), enzymes (ALT, AST, LDH, CK, ALP, ACP, amylase, lipase) and vitamins.

4. Red blood cells are employed for the determination of abnormal hemoglobins, glucose 6-phosphate dehydrogenase, pyruvate kinase etc.

Collection and preservation of blood specimens

Lack of thought before collecting specimens or carelessness in collection may adversely affect the interpretation or impair the validity of the tests carried out on the specimens. Some factors to consider include the following:

1. Diet Dietary constituents may alter the concentrations of analytes in blood significantly (e.g. plasma [glucose] and [triglyceride] are affected by carbohydrate and fat-containing meals, respectively).

2. Drugs Many drugs influence the chemical composition of blood. Such effects of drug treatment, for example, antiepileptic drugs, have to be taken into account when interpreting test results. Details of relevant drug treatment must be given when request­ing chemical analyses, especially when toxicological investigations are to be performed.

3. Diurnal variation. The concentrations of many substances in blood vary considerably at different times of day (e.g. cortisol). Specimens for these analyses must be collected at the times specified by the laboratory, as there may be no reference ranges relating to their concentrations in blood at other times


Care when collection blood specimens

The posture of the patient, the choice of skin-cleansing agent and the selection of a suitable vien (or other source) are the principal factors to consider before proceeding to collect each specimen:

1.  The skin must be clean over the site for collecting the blood specimen. However, it must be remembered that alconol and methylated spirits can cause haemolysis, and that their use is clearly to be avoided if blood [ethanol] is to be determined.

2.  Limbs into which intravenous infusions are being given must not be selected as the site of venepuncture unless particular care is taken. The needle or cannula must first be thoroughly flushed out with blood to avoid dilution of the specimen with infusion fluid.

3.  Venepuncture technique should be standardised as far as possible to enable closer comparison of successive results on patients.

4. Venous blood specimens should be obtained with minimal stasis Prolonged stasis can markedly raise the concentrations of plasma proteins and other non-diffusible substances (e.g. protein-bound substances). It is advisable to release the tourniquet before withdrawing the sample of blood.

5. Posture should be standardised if possible When a patient's posture changes from lying to standing, there may be an increase of as much as 13% in the concentration of plasma proteins or protein-bound constituents, due to redistribution of fluid in the extracellular space.

6.  Haemolysis should be avoided, since it renders specimens unsuitable for plasma K+, magnesium and many protein and enzyme activity measurements.

7. Infection hazard  High-risk specimens require special care in collection, and this danger must be clearly indicated on the request form.

 Vacutainers used for blood collection and storage

Care of blood specimens after collection

Blood specimens should be transported to the laboratory as soon as possible after collection. Special arrangements are needed for some specimens (e.g. for acid-base measurements, or unstable hormones) because of their lack of stability. Most other analytes are stable for at least 3 h in whole blood, or longer if plasma or serum is first separated from the cells. As a rule, whole blood specimens for chemical analysis must not be stored in a refrigerator, since ionic pumps that maintain electrolyte gradients across the cell membrane are inactive at low temperatures. Conversely, separated serum or plasma is best refrigerated, to minimize chemical changes or bacterial growth.

Several changes occur in whole blood specimens following collection. The commoner and more important changes that occur prior to the separation of plasma or serum from the cells are:

1.  Glucose is converted to lactate: this process is inhibited by fluoride;

2.  Several substances pass through the erythrocyte membrane, or may be added in significant amounts to plasma as a result of red cell destruction insufficient to cause detectable haemolysis. Examples include K+ and lactate dehydrogenize;

3. Loss of CO2 occurs, since the Pco2, of blood is much higher than in air;

4. Plasma [phosphate] increases due to hydrolysis of organic ester phosphates in the red cells;

5. Labile plasma enzymes lose their activity.



Certain biochemical tests require unclotted blood. Serum from coagulated blood is the specimen of choice for many assay systems.

Heparin (inhibits the convension prothrmobin to thrombin) is the most widely used anticoagulant for clinical chemical analysis. Heparin is an ideal anticoagulant, since it does not cause any change in blood composition. However, other anticoagulants are prefered to heparin, due to the cost factor.

Ethylene diamine tetra acetic acid (EDTA) is a chelating agent, and is  particularly useful for hematological examination because it preserves cellular components of the blood. It chelates with calcium and blocks coagulation. EDTA is employed to collect blood for hematological examinations It may affect some of the clinical chemistry tests.

Sodium fluoride is usually used as a preservative for blood glucose by inhibiting the enzyme systems involved in the glycolysis. Without an antiglycolytic agent, the blood glucose concentration decreases about 10 mg/dl per hour and false results may be obtained. Fluoride is also anticoagulant. It should not be used for enzyme assays, as well as when the test involves enzymatic analysis.

Citrate is widely used for coagulation studies.

Oxalate inhibits blood coagulation by forming insoluble complexes with calcium ions. Potassium oxalate may be used at a concentration of 1 -2 mg/ml blood. At concentration of > 3   mg/ml, oxalate may cause hemolysis.  

Potassium or sodium oxalate : These compounds precipitate calcium and inhibit blood coagulation. Being more soluble, potassium oxalate (5-10 mg per 5 ml blood) is prefered.

Potasium oxalate and sodium fluoride : These anticoagulants are employed for collecting blood to estimate glucose. Further sodium fluoride inhibits glycolysis and preserves bfood glucose concentration.

Ammonium oxalate and potassium oxalate : A  mixture of these two compounds in the ratio

 3 : 2 is used for blood collection to carry out certain hematological tests.


The rupture or lysis of RBC, releasing the cellular constituents interferes with the laboratory investigations. Therefore, utmost care should be taken to avoid hemolysis when plasma or serum are used for biochemical tests. Use of dry syringes, needles and containers, allowing slow flow of blood into syringe are among the important precautions to avoid hemolysis.


Plasma or serum should be separated within 2 hours after blood collection. It is ideal and advisable to analyse blood, plasma or serum, immediately after the specimen collection. This however, may not be always possible. In such a case, the samples (usually plasma/serum) can be stored at 4°C until analysed. For enzyme analysis, the sample are preserved at -20°C.

Sampling Errors:

1. Blood sampling technique. Difficulty in obtaining a blood specimen may lead to haemolysis with consequent release  of potassium and other red cell constituents. Results for these will be falsely elevated.

2. Prolonged stasis during venepuncture.  Plasma water diffuses into the interstitial space and the serum or plasma sample obtained will be concentrated. Proteins and protein-bound components of plasma such calcium or thyroxine will be falsely elevated.

3. Insufficient specimen. Each biochemical analysis requires a certain volume of specimen to enable  the test to be carried out.

4. Errors in timing. The biggest source of error in the measurement of any analyte in a 24-hour urine specimen is in the collection of an accurately timed volume of urine.

5. Incorrect specimen container. For many analyses the blood must be collected into a container with anticoagulant and preservative. For example, samples for glucose should be collected into a special container containing fluoride which inhibits glycolysis; otherwisethe time taken to deliver the sample to the laboratory can affect the result.

6. Inappropriate sampling site. Blood samples should not be taking downstream from an intravenous drip. It is not unheard of for the laboratory to receive a blood glucose request on a specimen taken from the same arm into which 5% glucose is being infused.

7. Incorect specimen storage. A blood sample stored overnight before being sent to the laboratory will show falsely high potassium, phosphate and red cells enzymes such as lactate dehydrogenize, because of leakage into the extracellular fluid from the cells.

          Many hormones show circardian rhythm. For example, ACTH has maximum peak at early morning, and minimum level at afternoon. Maximum level of growth hormone is during night and minimum is in the day time. Many  reference values are age related; e.g., levels of urea and cholesterol are more in geriatric patients. Exercise will increase the level of transaminases and creatinine. Triglyceride level is to be done in fasting condition. Caffeine (coffee and tea) will increase the levels of free fatty acid, glycerol, total lipids and glucose. Smoking will increase the levels of GH, cortisol and triglycerides.

Blood constitutes 6 to 8 percent of total body weight. In terms of volume, women have 4.5 to 5.5 L of blood and men 5 to 6 L. In infants and children, blood volume is 50 to 75 mL/kg in girls and 52 to 83 mL/kg in boys. The principal functions of blood are the transport of oxygen, nutrients, and hormones to all tissues and the removal of metabolic wastes to the organs of excretion. Additional functions of blood are (1) regulation of temperature by transfer of heat to the skin for dissipation by radiation and convection, (2) regulation of the pH of body fluids through the buffer systems and facilitation of excretion of acids and bases, and (3) defense against infection by transportation of antibodies and other substances as needed. Blood consists of a fluid portion, called plasma, and a solid portion that includes red blood cells (erythrocytes), white blood cells (leukocytes), and platelets (thrombocytes). Plasma makes up 45 to 60 percent of blood volume and is composed of water (90 percent), amino acids, proteins, carbohydrates, lipids, vitamins, hormones, electrolytes, and cellular wastes.



Hematology is traditionally limited to the study of the cellular elements of the blood, the production of these elements, and the physiological derangements that affect their functions.




Hemopoiesis – the processes of blood cells formation and development. 

There are 2 kinds of hemopoiesis: embrional and postembrional. Organs of embrional hemopoiesis: 1. In the first few weeks of gestation the yolk sac is the main site of hemopoiesis.2. Liver. 3. Spleen. 4. Lymphatic nodules. 5. Thymus. 6. Bone marrow – on 6-th month is central site of hemopoiesis. Organs of postembrional hemopoiesis: 1. Bone marrow (vertebrae, ribs, sternum, skull, sacrum and pelvis, proximal ends of femur). 2. Spleen. 3. Lymphatic nodules.

All blood cells derive from a common stem cell.



Under the influences of local and humoral factors, stem cells differentiate into different cell lines. Erythropoiesis and thrombopoiesis proceed independently once the stem cell stage has been passed, whereas monocytopoiesis and granulocytopoiesis are quite closely “related.” Lymphocytopoiesis is the most independent among the remaining cell series. Granulocytes, monocytes, and lymphocytes are collectively called leukocytes (white blood cells), a term that has been retained since the days before stainingmethods were available, when the only distinction that could be made was between erythrocytes (red blood cells) and the rest. All these cells are eukaryotic, that is, they are made up of a nucleus, sometimes with visible nucleoli, surrounded by cytoplasm, which may include various kinds of organelles, granulations, and vacuoles. Despite the common origin of all the cells, ordinary light microscopy reveals fundamental and characteristic differences in the nuclear chromatin structure in the different cell series and their various stages of maturation.


The developing cells in the granulocyte series (myeloblasts and promyelocytes), for example, show a delicate, fine “net-like” (reticular) structure. Careful microscopic examination (using fine focus adjustment to view different depth levels) reveals a detailed nuclear structure that resembles fine or coarse gravel. With progressive stages of nuclear maturation in this series (myelocytes, metamyelocytes, and band or staff cells), the chromatin condenses into bands or streaks, giving the nucleus— which at the same time is adopting a characteristic curved shape—a spotted and striped pattern. Lymphocytes, on the other hand—particularly in their circulating forms—always have large, solid-looking nuclei. Like cross-sections through geological slate, homogeneous, dense chromatin bands alternate with lighter interruptions and fissures. Each of these cell series contains precursors that can divide (blast precursors) and mature or almostmature forms that can no longer divide; the morphological differences between these correspond not to steps in mitosis, but result from continuous “maturation processes” of the cell nucleus and cytoplasm. Once this is understood, it becomes easier not to be too rigid about morphological distinctions between certain cell stages. The blastic precursors usually reside in the hematopoietic organs (bone marrow and lymph nodes). Since, however, a strict blood–bone marrow barrier does not exist (blasts are kept out of the bloodstream essentially only by their limited plasticity, i.e., their inability to cross the diffusion barrier into the bloodstream), it is in principle possible for any cell type to be found in peripheral blood, and when cell production is increased, the statistical frequency with which they cross into the bloodstream will naturally rise as well. Conventionally, cells are sorted left to right from immature to mature, so an increased level of immature cells in the bloodstream causes a “left shift” in the composition of a cell series—although it must be said that only in the precursor stages of granulopoiesis are the cell morphologies sufficiently distinct for this left shift to show up clearly.



Blood Cell functions.


Neutrophil granulocytes with segmented nuclei serve mostly to defend against bacteria. Predominantly outside the vascular system, in “inflamed” tissue, they phagocytose and lyse bacteria. The blood merely transports the granulocytes to their site of action.

The function of eosinophilic granulocytes is defense against parasites; they have a direct cytotoxic action on parasites and their eggs and larvae. They also play a role in the down-regulation of anaphylactic shock reactions and autoimmune responses, thus controlling the influence of basophilic cells.

The main function of basophilic granulocytes and their tissue-bound equivalents (tissue mast cells) is to regulate circulation through the release of substances such as histamine, serotonin, and heparin. These tissue hormones increase vascular permeability at the site of various local antigen activity and thus regulate the influx of the other inflammatory cells.

The main function of monocytes is the defense against bacteria, fungi, viruses, and foreign bodies. Defensive activities take place mostly outside the vessels by phagocytosis. Monocytes also break down endogenous cells (e.g., erythrocytes) at the end of their life cycles, and they are assumed to perform a similar function in defense against tumors. Outside the bloodstream, monocytes develop into histiocytes; macrophages in theendothelium of the body cavities; epithelioid cells; foreign body macrophages (including Langhans’ giant cells); and many other cells.

Lymphocytes are divided into two major basic groups according to function. Thymus-dependent T-lymphocytes, which make up about 70% of lymphocytes, provide local defense against antigens fromorganic and inorganic foreign bodies in the form of delayed-type hypersensitivity, as classically exemplified by the tuberculin reaction. T-lymphocytes are divided into helper cells and suppressor cells. The small group of NK (natural killer) cells, which have a direct cytotoxic function, is closely related to the T-cell group.

The other group is the bone-marrow-dependent B-lymphocytes or Bcells, which make up about 20% of lymphocytes. Through their development into immunoglobulin-secreting plasma cells, B-lymphocytes are responsible for the entire humoral side of defense against viruses, bacteria, and allergens. Erythrocytes are the oxygen carriers for all oxygen-dependent metabolic reactions in the organism. They are the only blood cells without nuclei, since this allows them to bind and exchange the greatest number of O2 molecules. Their physiological biconcave disk shape with a thick rim  provides optimal plasticity.

Thrombocytes form the aggregates that, along with humoral coagulation factors, close up vascular lesions. During the aggregation process, in addition to the mechanical function, thrombocytic granules also release  factors that promote coagulation. Thrombocytes develop from polyploid megakaryocytes in the bone marrow. They are the enucleated, fragmented cytoplasmic portions of these progenitor cells.



A CBC includes (1) enumeration of the cellular elements of the blood, (2) evaluation of RBC indices, and (3) determination of cell morphology by means of stained smears.


Because the CBC provides much information about the overall health of the individual, it is an essential component of a complete physical examination,

especially when performed on admission to a health-care facility or before surgery. Other indications for a CBC are as follows:

1.    Suspected hematologic disorder, neoplasm, or immunologic abnormality

2.    History of hereditary hematologic abnormality

3.    Suspected infection (local or systemic, acute or chronic)

4.    Monitoring effects of physical or emotional stress

5.    Monitoring desired responses to drug therapy and undesired reactions to drugs that may cause blood dyscrasias

6.    Monitoring progression of nonhematologic disorders such as chronic obstructive pulmonary disease, malabsorption syndromes, malignancies, and renal disease

Taking Blood Samples

This means that blood should always be drawn at about the same time of day and after at least eight hours of fasting, since both circadian rhythm and nutritional status can affect the findings. If strictly comparable values are required, there should also be half an hour of bed rest before the sample is drawn, but this is only practicable in a hospital setting. In other settings (i.e., outpatient clinics), bringing portable instruments to the relaxed, seated patient works well.

A sample of capillary blood may be taken when there are no further tests that would require venous access for a larger sample volume. A well perfused fingertip or an earlobe is ideal; in newborns or young infants, the heel is also a good site. If the circulation is poor, the blood flow can be increased by warming the extremity by immersing it in warm water. Without pressure, the puncture area is swabbed several times with 70% alcohol, and the skin is then punctured firmly but gently with a sterile disposable lancet. The first droplet of blood is discarded because it may be contaminated, and the ensuing blood is drawn into the pipette (see below). Care should be taken not to exert pressure on the tissue from which the blood is being drawn, because this too can change the cell composition of the sample.

General blood analysis (normal values)

1.    Erythrocytes (red blood cells) Male - 4-5,1× 1012/L

                                              Female – 3,7-4,7× 1012/L

2.    Hemoglobin    Male - 130-160 g/L    Female – 120-140 g/L   

3.    Hematocrit                   Male - 40-48 %    Female – 36-42 %

4.    Reticulocytes                       0,5-1 %

5.    Plateletes                              180-320 × 109/L

6.    ESR (Erythrocytes sedimentation rate)  Male - 1-10 mm/hour

                                                              Female – 2-15 mm/hour

7.    Leucocytes 4-9 × 109/L

Neutrophilic band granulocytes – 1-6 %

Neutrophilic segmented granulocytes – 45-72 %

Eosinophilic granulocytes – 0,5-5 %

Basophilic granulocytes – 0-1 %

Monocytes – 3-11 %

Lymphocytes – 19-37 %


Erythrocyte Parameters

The quality of erythrocytes is characterized by:


1.    MCV (mean corpuscular volume)

Male - 80-94 mcm3 (Fl) Female – 81-99  mcm3 (Fl)

2.    MCH (mean corpuscular hemoglobin) – 27-31 pg

3.    MCHC (mean corpuscular hemoglobin concentration) – 33-37 % or 20,4-22,9 mmol/L

4.    Red Cell Distribution Width (RDW) - 11,5-14,5 %.



MCV indicates the volume of the Hgb in each RBC, MCH is the weight of the

Hgb in each RBC, and MCHC is the proportion of Hgb contained in each RBC. MCHC is a valuable indicator of Hgb deficiency and of the oxygen-carrying

capacity of the individual erythrocyte. A cell of abnormal size, abnormal shape, or both may contain an inadequate proportion of Hgb. RBC indices are used mainly in identifying and classifying types of anemias. Anemias are generally classified according to RBC size and Hgb content. Cell size is indicated by the terms normocytic, microcytic, and macrocytic. Hemoglobin content is indicated by the terms normochromic, hypochromic, and hyperchromic.




The erythrocyte (RBC) count, a component of the CBC, is the determination of the number of RBCs per cubic millimeter. In international units, this is expressed as the number of RBCs per liter of blood. The test is less significant by itself than it is in computing Hgb, Hct, and RBC indices. Many factors influence the level of circulating erythrocytes. Decreased numbers are seen in disorders involving impaired erythropoiesis excessive blood cell destruction (e.g., hemolytic anemia), and blood loss, and in chronic inflammatory diseases. A relative decrease also may be seen in situations with increased body fluid in the presence of a normal number of RBCs (e.g., pregnancy). Increases in the RBC count are most commonly seen in polycythemia vera, chronic pulmonary disease with hypoxia and secondary polycythemia, and dehydration with hemoconcentration. Excessive exercise,

anxiety, and pain also produce higher RBC counts.



Blood consists of a fluid portion (plasma) and a solid portion that includes RBCs, WBCs, and platelets. More than 99 percent of the total blood cell mass is composed of RBCs. The Hct or packed RBC volume measures the proportion of RBCs in a volume of whole blood and is expressed as a percentage. Several methods can be used to perform the test. In the classic method, anticoagulated venous blood is pipetted into a tube 100 mm long and then centrifuged for 30 minutes so that the plasma and blood cells separate. The volumes of packed RBCs and plasma are read directly from the millimeter marks along the side of the tube. In the micromethod, venous or capillary blood is used to fill a small capillary tube, which is then centrifuged for 4 to 5 minutes. The proportions of plasma and RBCs are determined by means of a calibrated reading device. Both techniques allow visual estimation of the volume of WBCs and platelets. With the newer, automated methods of cell counting, the Hct is calculated indirectly as the product of the RBC count and mean cell volume. Although this method is generally quite accurate, certain clinical situations may cause errors in interpreting the Hct. Abnormalities in RBC size and extremely elevated WBC counts may produce false Hct values. Elevated blood glucose and sodium may produce elevated Hct values because of the resultant swelling of the erythrocyte. Normally, the Hct parallels the RBC count. Thus, factors influencing the RBC count also affect the results of the Hct.




Hemoglobin is the main intracellular protein of the RBC. Its primary function is to transport oxygen to the cells and to remove carbon dioxide from them for excretion by the lungs. The Hgb molecule consists of two main components: heme and globin. Heme is composed of the red pigment porphyrin and iron, which is capable of combining loosely with oxygen. Globin is a protein that consists of nearly 600 amino acids organized into four polypeptide chains. Each chain of globin is associated with a heme group. Each RBC contains approximately 250 million molecules of hemoglobin, with some erythrocytes containing more hemoglobin than others. The oxygen-binding, -carrying, and –releasing capacity of Hgb depends on the ability of the globin chains to shift position normally during the oxygenation–deoxygenation process. Structurally abnormal chains that are unable to shift normally have decreased oxygen-carrying ability. This decreased oxygen transport capacity is characteristic of anemia. Hemoglobin also functions as a buffer in the maintenance of acid–base balance. During transport, carbon dioxide (CO2) reacts with water (H2O) to form carbonic acid (H2CO3). This reaction is speeded by carbonic anhydrase, an enzyme contained in RBCs. The carbonic acid rapidly dissociates to form hydrogen ions (H+) and bicarbonate ions (HCO3). The hydrogen ions combine with the Hgb molecule, thus preventing a buildup of hydrogen ions in the blood. The bicarbonate ions diffuse into the plasma and play a role in the bicarbonate buffer system. As bicarbonate ions enter the bloodstream, chloride ions (Cl_) are repelled and move back into the erythrocyte. This “chloride shift” maintains the electrical balance between RBCs and plasma.

Hemoglobin determinations are of greatest use in the evaluation of anemia, because the oxygen-carrying capacity of the blood is directly related to the Hgb level rather than to the number of erythrocytes. To interpret results accurately, the Hgb level must be determined in combination with the Hct level. Normally, Hgb and Hct levels parallel each other and are commonly used together to express the degree of anemia. The combined values are also useful in evaluating situations involving blood loss and related treatment. The Hct level is normally three times the Hgb level. If erythrocytes are abnormal in shape or size or if Hgb manufacture is defective, the relationship between Hgb and Hct is disproportionate.





The stained RBC examination (RBC morphology) involves examination of RBCs under a microscope. It is usually performed to compare the actual appearance of the cells with the calculated values for RBC indices. Cells are examined for abnormalities in color, size, shape, and contents. The test is performed by spreading a drop of fresh anticoagulated blood on a glass slide. The addition of stain to the specimen is used to enhance RBC characteristics.


Red Blood Cell Abnormalities Seen on Stained Smear



Descriptive Term






Cell diameter > 8 µm

MCV > 95 µm3


Megaloblastic anemias

Severe liver disease




Cell diameter < 6 µm

MCV < 80 µm3

MCHC< 27


Iron-deficiency anemia


Anemia of chronic disease



Increased zone of central pallor

Diminished Hgb content




Microcytic, hyperchromic cells

Increased bone marrow stores of iron


Chronic inflammation

Defect in ability to use iron for Hgb synthesis




Presence of red cells not fully






Variability of cell shape


Sickle cell disease

Microangiopathic hemolysis


Extramedullary hematopoiesis

Marrow stress of any cause



Red Blood Cell Abnormalities Seen on Stained Smear



Descriptive Term






Variability of cell size



Transfusing normal blood into microcytic or macrocytic cell population




Hypochromic cells with small

central zone of Hgb (“target




Obstructive jaundice




Cells with no central pallor,

loss of biconcave shape


Loss of membrane relative to cell volume

Hereditary spherocytosis




MCHC high


Accelerated red blood cell destruction by

reticuloendothelial system




Presence of cell fragments in



Increased intravascular mechanical trauma

Microangiopathic hemolysis




Irregularly spiculated surface

Regularly spiculated cell surface


Irreversibly abnormal membrane lipid content

Liver disease


Reversible abnormalities of membrane lipids

High plasma-free fatty acids

Bile acid abnormalities

Effects of barbiturates, salicylates, and so on




Elongated, slitlike zone of central



Hereditary defect in membrane sodium metabolism

Severe liver disease



Oval cells

Hereditary anomaly, usually harmless


Types of Abnormal Red Blood Cell Inclusions and Their Causes



Causes of inclusion

Heinz bodies (denatured Hgb)



G-6-PD deficiency

Hemolytic anemias



Drugs: analgesics, antimalarials, antipyretics, nitrofurantoin (Furadantin),

nitrofurazone (Furacin), phenylhydrazine, sulfonamides, tolbutamide,

vitamin K (large doses)


Basophilic stippling (residual

cytoplasmic RNA)


Anemia caused by liver disease

Lead poisoning



Howell-Jolly bodies (fragments

of residual DNA)



Intense or abnormal RBC production resulting from hemolysis or inefficient



Cabot’s rings (composition



Same as for Howell-Jolly bodies


Siderotic granules (ironcontaining


Abnormal iron metabolism

Abnormal hemoglobin manufacture




The osmotic fragility test determines the ability of the RCB membrane to resist rupturing in a hypotonic saline solution. Normal disk-shaped cells can imbibe water and swell significantly before membrane capacity is exceeded, but spherocytes (RBCs that lack the normal biconcave shape) and cells with damaged membranes burst in saline solutions only slightly less concentrated than normal saline. Conversely, in thalassemia, sickle cell disease, and other disorders. The test is performed by exposing RBCs to increasingly dilute saline solutions. The percentage of the solution at which the cells swell and rupture is then noted. Normal erythrocytes rupture in saline solutions of 0.30 to 0.45 percent. RBC rupture in solutions of greater than 0.50 percent saline indicates increased fragility. Lack of rupture in solutions of less than 0.30 percent saline indicates decreased RBC fragility.


Causes of Altered Erythrocyte Osmotic Fragility


Decreased Fragility


Increased Fragility

Iron-deficiency anemias


Hereditary spherocytosis


Hereditary anemias (sickle cell, hemoglobin C,



Hemolytic anemias



Liver diseases


Autoimmune anemias


Polycythemia vera






Toxins (bacterial, chemical)


Obstructive jaundice


Hypotonic infusions



Transfusion with incompatible blood



Mechanical trauma to RBCs (prosthetic heart valves,

disseminated intravascular clotting, parasites)



Enzyme deficiencies (PK kinase, G-6-PD




The erythrocyte sedimentation rate (ESR or sedrate) measures the rate at which RBCs in anticoagulated blood settle to the bottom of a calibrated tube. In normal blood, relatively little settling occurs because the gravitational pull on the RBCs is almost balanced by the upward force exerted by the plasma. If plasma is extremely viscous or if cholesterol levels are very high, the upward trend may virtually

neutralize the downward pull on the RBCs. In contrast, anything that encourages RBCs to aggregate or stick together increases the rate of settling. Inflammatory and necrotic processes, for example, cause an alteration in blood proteins that results in

clumping together of RBCs because of surface attraction. These clumps are called rouleaux. If the proportion of globin to albumin increases or if fibrinogen 3 levels are especially high, rouleaux formation is enhanced and the sed rate increases.



Causes of Altered Erythrocyte Sedimentation Rates


Increased rate

Decreased rate

Pregnancy (uterine and ectopic)

Polycythemia vera


Toxemia of pregnancy

Congestive heart failure


Collagen disorders (immune disorders of connective


Sickle cell, Hgb C disease


Inflammatory disorders

Degenerative joint disease





Acute myocardial infarction

Drug toxicity (salicylates, quinine derivatives,

adrenal corticosteroids

Most malignancies


Drugs (oral contraceptives, dextran, penicillamine,

methyldopa, procainamide, theophylline, vitamin A)


Severe anemias


Myeloproliferative disorders


Renal disease (nephritis)


Hepatic cirrhosis


Thyroid disorders


Acute heavy metal poisoning







Anemia may be defined as any condition resulting from a significant decrease in the total body erythrocyte mass. Measurement of total body rbc mass requires special radiolabeling techniques that are not amenable to general medical diagnostic work. Measurements typically substituted for rbc mass determination take advantage of the body's tendency to maintain normal total blood volume by dilution of the depleted rbc component with plasma. This adjustment results in decrease of the total blood hemoglobin concentration, the rbc count, and the hematocrit. Therefore, a pragmatic definition of anemia is a state which exists when the hemoglobin is less than 12 g/dL or the hematocrit is less than 37 cL/L. Anemia may exist as a laboratory finding in a subjectively healthy individual, because the body can, within limits, compensate for the decreased red cell mass.

One must be careful in blindly applying this practical definition of anemia in every case. As the following diagram shows, it is possible to be severely anemic and have a normal hematocrit (and hemoglobin). This occurs when there is rapid hemorrhage, with red cells and plasma being rapidly lost simultaneously, before the body can respond by hiking up the plasma volume.

Red cell and plasma volume

The final example in the above diagram illustrates that a person can have a low hematocrit and not be anemic. This occurs when a patient is overhydrated, typically as a result of overenthusiastic intravenous fluid therapy.

Clinical signs and symptoms of anemia

When the above mechanisms are overwhelmed by the increasing magnitude of the anemia, or when the demands of physical activity or intercurrent illness overwhelm them, a clinical disease state becomes apparent to the physician and to the patient. The severity of clinical symptoms bears less relationship to the severity of the anemia than to the length of time over which the condition develops. An acute hemorrhagic condition may produce symptoms with loss of as little as 20% of the total blood volume (or 20% of the total red cell mass). Conversely, anemias developing over periods long enough to allow compensatory mechanisms to operate will allow much greater loss of rbc mass before producing symptoms. It is not terribly uncommon to see a patient with a hemoglobin of 4 g/dL (hematocrit 12 cL/L), representing a loss of 70% of the rbc mass, being reluctantly dragged into a clinic by relatives concerned that he or she is looking a bit washed out.

When symptoms do develop, they are pretty much what you would expect given the precarious state of oxygen delivery to the tissues: dyspnea on exertion, easy fatigability, fainting, lightheadedness, tinnitus, and headache. In addition, the hyperdynamic state of the circulatory system can produce palpitations and roaring in the ears. Pre-existing cardiovascular pathologic conditions are, as you would expect, exacerbated by the anemia. Angina pectoris, intermittent claudication, and night muscle cramps speak to the effect of anemia on already compromised perfusion.

Clinical signs of a slowly developed anemia are pallor, tachycardia, and a systolic ejection murmur. In rapidly developing anemia (as from hemorrhage and certain catastrophic hemolytic anemias), additional symptoms and signs are noted: syncope on rising from bed, orthostatic hypotension (i.e., the blood pressure falls when the patient is raised from the supine to the sitting or standing positions) and orthostatic tachycardia. Keep in mind that if anemia develops through rapid enough bleeding, the hematocrit and hemoglobin will be normal (since in hemorrhage the rbc's and plasma are lost in proportion). Because of this, your appreciation of these clinical signs will serve you better in diagnosing this type of anemia than will the laboratory.

Classification of anemias

Anemias can be classified by cytometric schemes (i.e., those that depend on cell size and hemoglobin-content parameters, such as MCV and MCHC), erythrokinetic schemes (those that take into account the rates of rbc production and destruction), and biochemical/molecular schemes (those that consider the etiology of the anemia at the molecular level.

An example: sickle cell anemia

  • Cytometric classification: normochromic, normocytic
  • Erythrokinetic classification: hemolytic
  • Biochemical/molecular classification: DNA point mutation producing amino acid substitution in hemoglobin beta chain

A. Cytometric classification

Because cytometric parameters are more easily and less expensively measured than are erythrokinetic and biochemical ones, it is most practical to work from the cytometric classification, to the erythrokinetic, and then (hopefully) to the biochemical. Your first job in working up a patient with anemia is to place the case in one of three major cytometric categories:

1.     Normochromic, normocytic anemia (normal MCHC, normal MCV).

These include:

1.     anemias of chronic disease

2.     hemolytic anemias (those characterized by accelerated destruction of rbc's)

3.     anemia of acute hemorrhage

4.     aplastic anemias (those characterized by disappearance of rbc precursors from the marrow)

2.     Hypochromic, microcytic anemia (low MCHC, low MCV).

These include:

1.     iron deficiency anemia

2.     thalassemias

3.     anemia of chronic disease (rare cases)

3.     Normochromic, macrocytic anemia (normal MCHC, high MCV).

These include:

1.     vitamin B12 deficiency

2.     folate deficiency

B. Erythrokinetic classification

You would now want to proceed with classifying your case based on the rate of rbc turnover. If this is high, a normoregenerative anemia exists. Such anemias are seen in hemolysis (excess destruction of rbc's) or hemorrhage (loss of rbc's from the vascular compartment. In these cases, the marrow responds appropriately to anemia by briskly stepping up the production of rbc's and releasing them into the bloodstream prematurely. There are several lab tests that allow you to determine if increased rbc turnover exists:

1.     Reticulocyte count

A sample of blood is stained with a supravital dye that marks reticulocytes. An increased number of reticulocytes is seen when the marrow is churning out rbc's at excessive speed (presumably to make up for those lost to hemolysis or hemorrhage). Most labs will report the result of the reticulocyte count in percent of all rbc's counted. A typical normal range is 0.5-1.5 %. Making clinical decisions based on this raw count is somewhat fallacious.

For instance: A normal person with an rbc count of 5,000,000 /microliter and an absolute reticulocyte count of 50,000 /microliter would have a relative retic count of 1.0%. An anemic person with 2,000,000 rbc's/microliter and the same 50,000 retics/microliter would have an apparently "abnormal" relative retic count of 2.5 % and could be misdiagnosed as having high turnover.

Clearly, one needs to find some way to correct the raw retic count so as to avoid this problem. One can easily calculate the absolute retic count (in cells/microliter) by multiplying the rbc count by the relative retic count. The normal range for the absolute retic count is 50,000-90,000 /microliter.

2.     Bone marrow biopsy

This can be used to directly observe any accelerated production of rbc's. The ratio of the number of myeloid to erythroid precursors (the M:E ratio) tends to decrease in high-production states, and the marrow becomes hypercellular. Marrow biopsy is not usually performed just to measure the M:E ratio, but to answer other hematologic questions that have been raised.

The normoregenerative anemias are in contrast to those characterized by inadequate marrow response to the degree of anemia. These are the hyporegenerative anemias. In such cases, the reticulocyte production index is decreased. The classic example is aplastic anemia, in which there is primary marrow failure to produce enough erythrocyte mass. As you have probably come to expect, the distinction of these categories is not always absolute. For instance, in thalassemia major there is a degree of hemolysis (generally associated with the normoregenerative states) and inadequate marrow response to the degree of anemia.

C. Biochemical classification

Finally, one should attempt to determine the etiology of the anemia as specifically as possible. In some cases (e.g., iron deficiency), etiologic classification is easily attained; in others (e.g.. aplastic anemia) the biochemical mechanism of disease may be hopelessly elusive. Generally, biochemical tests are aimed at identifying a depleted cofactor necessary for normal hematopoiesis (iron, ferritin, folate, B12), an abnormally functioning enzyme (glucose-6-phosphate dehydrogenase, pyruvate kinase), or abnormal function of the immune system (the direct antiglobulin [Coombs'] test).

Nutritional Anemias And Anemia of Chronic Disease

Iron metabolism and iron deficiency anemia

A. Iron and its metabolism

The fourth most abundant element in the earth's crust, iron is only a trace element in biologic systems, making up only 0.004% of the body's mass. Yet it is an essential component or cofactor of numerous metabolic reactions. By weight, the great proportion of the body's iron is dedicated to its essential role as a structural component of hemoglobin. Hemoglobin without iron is totally useless (in fact, hemoglobin with Fe+++ instead of the normal Fe++ is the ugly brown methemoglobin and is also worthless as an oxygen carrier). Without sufficient iron available to the rbc precursors, normal erythropoiesis cannot take place, and anemia develops. On the other hand, iron is a toxic substance. Too much iron accumulating in vital structures (especially the heart, pancreas, and liver) produces a potentially fatal condition, hemochromatosis. Clearly, iron, like oxygen, is another of the deleterious substances that evolution has led biologic systems into flirtation with.

Most of the iron not circulating in the rbc's is stored in the Fe+++ (ferric) oxidation state. This iron is stored in marrow histiocytes in the form of hemosiderin. When iron is needed by the erythron, the hemosiderin gives up its iron to nearby rbc precursors who line up around the histiocyte like pigs around a trough. Hemosiderin is easy to see microscopically in smear or section preparations of marrow, due to the ferric iron's ability to produce an intense blue color in the Prussian blue stain. This reaction is the basis of the routine "iron stain" done on bone marrow specimensto assess adequacy of depot iron. Erythrocytes would not be expected to stain positively, since they contain ferrous iron. Because the body is dealing with such an essential but dangerous and biologically rare substance (and because you have become resigned to endless memorization in your hazing as medical students), you would expect that there would be some kind of complicated mechanism for the absorption and transport of iron.

Iron is present in greatest concentration in meat and dark green vegetables. The U.S. Recommended Daily Allowance for adults is 10 mg for males, 18 mg for menstruating females. The average daily American diet contains about 10 mg iron, of which only about 1 mg is absorbed. What goes in must come out, and in the adult male, the 1 mg/day iron loss occurs almost exclusively in the stool. For reproductive-aged females, an additional route is the menstrual flux, which accounts for a wildly variable incremental loss. While the average monthly menstrual blood loss is 40 mL (equivalent to 16 mg iron), some women who consider themselves healthy may lose up to 495 mL blood (about 200 mg iron) per menstrual period, or an average of about 7 mg iron per day (200 mg iron ÷ 28 days/cycle). It is not surprising that iron deficiency anemia is relatively common in women of this age group.

Following ingestion, iron is absorbed primarily in the duodenum, although any portion of the small bowel is efficient at iron absorption (in contrast to the situation with B12, as noted below). Only ferrous iron can be absorbed. The normal gastric acidity provides an optimal environment for the reduction of any ferric iron to the ferrous version. In states of iron depletion, a greater proportion of iron is absorbed than in states of normal iron depots. After uptake, the ferrous iron is transported to the subepithelial capillaries (possibly by intracellular transferrin), and released into the bloodstream. There it is oxidized to Fe+++ and again taken up by plasma transferrin. It is then conveyed to the erythron (and reduced again to the ferrous version) or to marrow histiocytes for eventual incorporation into hemoglobin. Storage iron exists as part of a ferric iron-apoprotein complex called ferritin. Ferritin molecules are water-soluble and are present in plasma in concentration equilibrium with ferritin molecules in histiocytes. Therefore, decreased iron stores (as is seen in impending iron deficiency anemia) are reflected by decreased serum concentration of ferritin, a substance easily measured in clinical laboratories. In marrow histiocytes, most of the ferritin molecules glom up into visible (through the microscope, that is) blobs of cytoplasmic inclusions rich in iron and poor in apoprotein; this substance is called hemosiderin. Hemosiderin is easily seen with the Prussian blue stain but can even be observed in unstained preparations of marrow, if present in sufficient quantities, due to the natural golden brown color of iron itself. Since hemosiderin is not soluble, it does not float around in the plasma with ferritin.

B. Iron deficiency anemia

When there is insufficient iron available for the normal production of hemoglobin, anemia results. The cells which are produced are small and pale, and indices from such specimens show low values for MCHC and MCV. Therefore, the classic anemia that occurs in iron deficiency is hypochromic, microcytic. Early or mild cases of iron deficiency anemia (IDA) show microcytosis without hypochromia. Since this is a hyporegenerative anemia, the retic count would be expected to be low; however, because so many cases of IDA are due to chronic bleeding, it is not uncommon to see patients with episodes of hemorrhage that have produced an elevated RPI on clinical presentation. It would appear that the marrow is able to produce a transient response to bleeding, but over the long haul it is a day late and a dollar short. Another finding commonly seen on clinical presentation is thrombocytosis, again probably reflecting marrow response to bleeding. The sine qua non of IDA is the observation that there is essentailly no iron in the marrow (that's zero, zilch, nada), since erythropoiesis can occur normally as long as at least some storage iron is present. In iron-deficient states, one of the body's clever reactive phenomena is the increase in production of transferrin. This is sometimes measured as total iron binding capacity of serum (TIBC). Without the availability if iron, a heme precursor, protoporphyrin, and a porphyrin side-reactant, zinc protoporphyrin, accumulate in the red cell. These may also be measured. In summary, the laboratory features of IDA are:

Hypochromic, microcytic anemia

Variable retic count

Increased erythrocyte zinc protoporphyrin

Increased free erythrocyte protoporphyrin

Decreased serum iron

Increased TIBC

Decreased serum ferritin

Absent marrow storage iron

Variable platelet count



Occurrence of macrocytes and microcytes

C. Causes of IDA

Iron stores can be depleted either through insufficient intake or excessive loss. In America, the combination of our meat-rich diet and fortification of our staples (such as Wonder Bread, Quaker Instant Grits, and Kellogg's 40% Bran Flakes) with added iron makes dietary insufficiency a very rare condition. The one exception to this is the case with milk-fed infants. Bovine milk has almost no iron. An iron-deficient state in such babies often is sown in the fertile soil of an antenatal life in a mother who was also overtly or borderline iron-deficient (iron requirements are markedly increased in pregnancy due to the demands of developing the fetal tissues). Fortunately most infant formulas are fortified with iron now. Moreover, today's parents are so paranoid about iron deficiency that it is surprising that the typical child of the 1990's can get through an airport without setting off any alarms. Still, nutritional cases of IDA do occur.

Although dietary deficiency of iron is rare, individuals with gastrointestinal lesions producing malabsorption syndromes may fail to assimilate sufficient iron to maintain the erythron, even in the face of adequate iron intake.

The much more important cause of iron depletion is chronic blood loss. In females, this is usually due to menses. Other more sinister causes include chronically bleeding lesions of the gastrointestinal tract, from reflux esophagitis, to peptic ulcers, to gastric or colorectal adenocarcinomas. Because these bad guys may be lurking asymptomatically, spilling erythrocytes here and there for months, all cases of iron deficiency anemia must be thoroughly investigated for the presence of bleeding sites. This is especially true in cases involving females who are not of reproductive age and in all males. In these demographic groups, to simply treat IDA with iron and not investigate for bleeding lesions is unequivocal gross negligence.

II. Anemia of Chronic Disease (ACD)

This is a condition seen in individuals suffering from chronic infections, noninfectious inflammatory diseases (such as rheumatoid arthritis), and neoplasms. The following pathogenetic observations have been made to help characterize the anemia:

  • Decreased rbc life span. This appears to be due to a factor or factors extrinsic to the red cell. The chemical nature of such factor(s) is completely unknown.
  • Impaired iron metabolism. Iron accumulates in the marrow histiocytes, but its uptake into rbc precursors is impaired. Therefore the marrow shows decreased sideroblastic iron in the face of increased histiocytic iron. This is probably because lactoferrin (an iron-containing compound made by neutrophils to employ in destroying bacteria) competes with transferrin for surface receptors on macrophages. The iron in lactoferrin is not available for use by developing red cell precursors.
  • Refractoriness to erythropoietin is an effect of lymphokines that are secreted by turned-on immune cells. This anti-growth effect of inflammation is not limited to erythroblasts; even hair and nails grow more slowly in times of inflammation.

The anemia is usually said to be normochromic/normocytic, but most patients actually have a slightly decreased MCHC (thus hypochromia). A minority of patients will be microcytic as well. The serum iron is decreased, as is the transferrin (or TIBC) in contrast to iron deficiency anemia, where transferrin is elevated. The absolute retic count is normal or slightly elevated. Bone marrow biopsy shows increased histiocytic iron and decreased sideroblastic iron, but no other morphologic findings are characteristic of this condition.

Megaloblastic anemias

These are a number of conditions which have in common the failure to synthesize adequate amounts of normal DNA. The anemias are macrocytic, since hemoglobinization is allowed, but cells mature more slowly in the marrow; therefore, the cells vegetate in the marrow, slowly maturing but stuffing their greedy little figurative mouths with iron, making hemoglobin, and getting larger as a result. Although some of these obese cells make it out of the marrow, many more never mature properly and eventually are destroyed before they have tasted the thrill of the extramedullary hunt. This phenomenon is referred to as ineffective erythropoiesis. Such marrows are packed with erythroid precursors, even in the face of severe anemia. The rbc precursors are notable morphologically for their immature, sometimes even blast-like chromatin in large nuclei. Such cells are called megaloblasts. Megaloblastic changes are not limited to the erythroid precursors, but are also seen in myeloid precursors. In some cases of megaloblastic anemia, there is concomitant leucopenia and thrombocytopenia, reflecting the troubled development of granulocytes and platelets as well.

A. Pathogenesis of megaloblastic anemias

The megaloblastic state results from an imbalance between supply of co-factors necessary for DNA synthesis and demand for DNA production. The two co-factors which are the most important are folate and vitamin B12. When these are deficient, megaloblastic change results. On the other hand, increased demand for DNA in physiologically hyperproliferative states, such as cancer and hemolytic anemia, can cause megaloblastic change even in the face of freely available folate and B12. To understand why folate and B12 are so important to DNA synthesis, it is necessary to gird one's loins for a trip back to Biochem. DNA differs from RNA in that 1) deoxyribose is used instead of ribose, and thymine is used instead of uracil. The structural formulas below show that uracil and thymine differ only by a silly little methyl group.

Structures of uracil and thymine

Unfortunately, the methyl is necessary for the magic DNA enzymes to recognize the molecule as DNA and work their wonders on the double helix. It seems that the body can make just about any organic molecule out of yesterday's BK Broiler and a few trace metals, but in this case a special set of reactions is necessary for the finishing touches on thymine. To stick the methyl group on the ring, folate is required, and B12 helps out.

B. Folate

Just to make casual observers think we're studying real science here, let us look at the chemical structure of folate and its metabolites.

Structures of folate congeners

Note that "folate" and "folic acid" are basically the same thing and differ only in whether the carboxylic acid groups are dissociated, this in turn dependent only on ambient pH. Folic acid is a vitamin found in abundance in many foods, especially asparagus, broccoli, endive, spinach, and lima beans. The daily requirement is only 50 µg (Cf. 10,000 µg for iron). The body's folate reserves last about four months. The vitamin is rapidly absorbed by the proximal jejunum. Folic acid is metabolically inactive until it is converted into tetrahydrofolic acid (THF). A key enzyme in this conversion is dihydrofolate reductase, which is the target enzyme inhibited by the anticancer drug methotrexate. THF is capable of methyl group transfer by picking up the one-carbon group from the amino acid serine and sticking it on uridylate, thus producing thymidylate, which in turn goes off to seek its fortune in DNA as a courier of genetic messages. The methyl-carrying version of THF is called N5,N10-THF and is shown above.

As complicated as all this seems, it only scratches the surface. Folate has been shown to play a role in no fewer than six biochemical reactions, including synthesis of methionine, synthesis of purines (thymine is a pyrimidine), and catabolism of histidine. Failure of folate to break down histidine results in accumulation of an intermediary metabolite, formiminoglutamic acid (FIGlu), which can be measured in the clinical laboratory as a marker for folate deficiency.

Deficiency of folate is seen among poorly nourished individuals, especially alcoholics, infants fed solely on milk, and pregnant women. Malabsorption syndromes often produce folate deficiency, and certain drugs (e.g., phenytoin, phenobarbital, primidone, isoniazid, and cycloserine) are associated with compromise of folate absorption and metabolism.

C. Vitamin B12

Vitamin B12 is a substance whose biochemistry is complex. Basically it consists of a cobalt-containing porphyrin-like prosthetic group attached to a nucleotide (cobalt is a major trace metal in human physiology, with the average body containing 1.1 grams of the element, third in abundance after iron and zinc). The diagram at left shows the structure of cyanocobalamin, one the simpler forms of the vitamin. B12 has been called the most complicated molecule in the human body that is not simply a repeating chain of smaller   units (like proteins and nucleic acids). The laboratory synthesis of this substance, not accomplished until 1971, capped off the distinguished career of Nobel-winning organosynthesist Robert Burns Woodward.

Deficiency of this vitamin produces megaloblastic anemia due to its role in folate metabolism. During the many transformations of folate from one form to another, a proportion gets accidentally converted to N5-methyl-THF, an inactive metabolite. This is called the "folate trap," since there is no way for active N5,N10-THF to be regenerated except through a reaction for which a form of vitamin B12, methyl-B12, is a cofactor (see diagram, right). Deficiency of B12 then produces a situation where more and more folate is trapped in an inactive form with no biochemical means of escape. The end result is failure to synthesize adequate DNA.

B12 deficiency also produces nervous system lesions not seen in folate deficiency. These lesions are manifest clinically as combined systems disease, a constellation of findings related to demyelination of axons in the spinal cord and cerebrum. These patients have decreased vibratory and proprioceptive senses in the extremities, spastic ataxia, disturbances of vision, taste, and smell, irritability, and somnolence. "Megaloblastic madness" is the term appended to the poorly documented cases of bizarre, sometimes psychotic behavior in B12-deficient patients. Clearly, there must be something that B12 is supposed to do about which folate has little or no concern. The best guess is that it has something to do with B12's role as a cofactor for methylmalonylCoA mutase, which catalyzes the following reaction:

Structures of uracil and thymine

A few of you, the truly perverted, will recognize this reaction as the clever way the body has of dealing with odd-chain fatty acids, prestidigitating them into even-chain species that can be handily dispatched via beta-oxidation. Without this mechanism, no telling what sorts of havoc these three-carbon residues would raise floating around in the myelin-making factory. A nice spin-off is that methylmalonic acid accumulates and shows up in the urine, where it can be measured as a marker of B12 deficiency.

The etiology of B12 deficiency is more complicated than that of folate deficiency. One can develop deficiency through either of the following mechanisms:

1.     Dietary deficiency

B12 is only found in animal products, but it is plentiful. Therefore, nutritional deficiency is seen almost exclusively in vegans. Even so, this is extremely rare. Unlike the situation with folate, B12 body reserves can last for years.

2.     Malabsorption states

By far, this is the most common mechanism of disease development. The absorption of B12 is much more complicated than that of folate and iron. B12 is absorbed only in the terminal ileum. Absorption occurs only if the B12 is bound to a glycoprotein, unimaginatively named intrinsic factor, produced only by the parietal cells of the gastric mucosa. Therefore, malabsorption can occur if 1) intrinsic factor is not produced, 2) intrinsic factor is neutralized, 3) there is a pathologic lesion of the terminal ileum, or 4) there are microorganisms present which compete successfully with the host for the B12. Let us consider each mechanism in turn:

o        Intrinsic factor not produced

This is seen following total gastrectomy. One must always give maintenance parenteral B12 for life following gastrectomy. Intrinsic factor is also not produced in pernicious anemia (see below).

o        Intrinsic factor inhibited

Pernicious anemia (PA) is the classic term used to describe the megaloblastic anemia which develops as a result of autoimmune destruction of the gastric mucosa (atrophic gastritis) and autoantibodies directed against intrinsic factor. PA is said to occur most commonly in elderly Caucasians of northern European extraction and in younger African-Americans. There is a strong association with other autoimmune diseases in the same patient, especially Hashimoto's thyroiditis.

o        Terminal ileum lesions

Crohn's disease is a classic example. It typically affects the terminal ileum and can produce malabsorption because of massive tissue destruction in this area.

o        Competition for B12

This occurs in various congenital or acquired anatomic abnormalities of the small intestine which foster overgrowth of our tiny little prokaryotic friends, all too happy to slurp up all that B12 at our expense. Another condition (a classic National Board question if there ever was one) is infestation with the fish tapeworm, Diphyllobothrium latum, also quite capable of quaffing a few cobalts.


Acute posthemorrhagic anemia

 or acute blood loss anemia - a disease associated with loss of a large volume of hemoglobin.

Etiology. Developed as a result of acute blood loss during injury or disease complicated with hemorrhage.

Symptoms and flow. Actually anemia with concomitant hypoxia, hemodynamic symptoms (collapse). Immediately after bleeding red blood is usually not sharply reduced due to a reflex decrease in total vascular channel and compensatory revenues deposited into the circulation of blood. After 1-2 days, upon receipt of a blood flow of tissue fluid and restore the original volume of the vascular channel, there is a uniform decline in hemoglobin and red blood cells. This anemia is classified as normochromic, normocytic, regenerative.

After 4-5 days, there are signs of regeneration of blood: reticulocytosis, neutrophilic leukocytosis with a shift of leukocyte counts to myelocytes and mild thrombocytosis. In bone marrow is determined by the increase in red growth of 30-40% (normal 16-20%) with predominance in it oxyphilic erythroblasts and normoblasts.

Recognition in most cases is not difficult. Difficulties arise when suddenly fledged internal bleeding (eg, rupture of fetal-receptacle with ectopic pregnancy).

Treatment. Immediate removal of the causes of bleeding, transfusion of at least 500 ml of whole blood or 250 ml of red blood cells. In the case of shock introduction substituting liquids: plasma or other blood substitutes.


Aplastic Anemia (Hypoplastic Anemia) anemia is a normocytic-normochromic anemia that results from a loss of blood cell precursors, causing hypoplasia of bone marrow, RBCs, WBCs, and platelets. Symptoms result from severe anemia, thrombocytopenia (petechiae, bleeding), or leukopenia (infections). Diagnosis requires demonstration of peripheral pancytopenia and the absence of cell precursors in bone marrow. Treatment is equine antithymocyte globulin and cyclosporine. Erythropoietin, granulocyte-macrophage colony-stimulating factor, and bone marrow transplantation may also be useful.

The term aplastic anemia commonly implies a panhypoplasia of the marrow with associated leukopenia and thrombocytopenia. In contrast, pure RBC aplasia is restricted to the erythroid cell line. Although both disorders are uncommon, aplastic anemia is more common.


True aplastic anemia (most common in adolescents and young adults) is idiopathic in about ½ of cases. Recognized causes are chemicals (eg, benzene, inorganic arsenic), radiation, and drugs (eg, antineoplastic drugs, antibiotics, NSAIDs, anticonvulsants, acetazolamide , gold salts, penicillamine , quinacrine). The mechanism is unknown, but selective (perhaps genetic) hypersensitivity appears to be the basis.

Fanconi's anemia is a very rare, familial form of aplastic anemia with bone abnormalities, microcephaly, hypogonadism, and brown pigmentation of skin. It occurs in children with abnormal chromosomes. Fanconi's anemia is often inapparent until some illness (especially an acute infection or inflammatory disorder) supervenes, causing peripheral cytopenias. With clearing of the supervening illness, peripheral values return to normal despite reduced marrow mass.

Pure RBC aplasia may be acute and reversible. Acute erythroblastopenia is a brief disappearance of RBC precursors from the marrow during various acute viral illnesses (particularly human parvovirus infection), especially in children. The anemia lasts longer than the acute infection. Chronic pure RBC aplasia has been associated with hemolytic disorders, thymomas, and autoimmune mechanisms and, less often, with drugs (eg, tranquilizers, anticonvulsants), toxins (organic phosphates), riboflavin deficiency, and chronic lymphocytic leukemia. A rare congenital form, Diamond-Blackfan anemia, usually occurs during infancy but has also been reported in adulthood. Diamond-Blackfan anemia is associated with bony abnormalities of the thumbs or digits and short stature.

Symptoms and Signs

Although onset of aplastic anemia usually is insidious, often occurring over weeks or months after exposure to a toxin, occasionally it is acute. Signs vary with the severity of the pancytopenia. Symptoms and signs of anemia (eg, pallor) usually are severe.

Severe thrombocytopenia may cause petechiae, ecchymosis, and bleeding from the gums, into the conjunctivae, or other tissues. Agranulocytosis commonly causes life-threatening infections. Splenomegaly is absent unless induced by transfusion hemosiderosis. Symptoms of pure RBC aplasia are generally milder and relate to the degree of the anemia or to the underlying disorder.


·                     CBC

·                     Bone marrow examination

Aplastic anemia is suspected in patients, particularly young patients, with pancytopenia (eg, WBC < 1500/μL, platelets < 50,000/μL). Pure RBC aplasia (including Diamond-Blackfan anemia) is suspected in patients with bony abnormalities and normocytic anemia but normal WBC and platelet counts. If either diagnosis is suspected, bone marrow examination is done.

In aplastic anemia, RBCs are normochromic-normocytic (sometimes marginally macrocytic). The WBC count reduction occurs chiefly in the granulocytes. Platelets are often far below 50,000/μL. Reticulocytes are decreased or absent. Serum iron is elevated. The bone marrow is acellular. In pure RBC aplasia, normocytic anemia, reticulocytopenia, and elevated serum iron are present, but with normal WBC and platelet counts. Bone marrow cellularity and maturation may be normal except for absence of erythroid precursors.


Hemolytic Anemias

Previously we have looked at nutritional anemias and the anemia of chronic disease, in which the metabolic needs of erythrocyte development are not met. The result is failure to produce enough healthy red cells. Now we turn to conditions in which the erythrocyte construction industry is healthy, but where the red cells produced are incapable of surviving the normal 120-day life span. These hemolytic anemias may be due to either intrinsic defects in rbc structure/function or a hostile external environment in which the cells are forced to live. To start with a few definitions:

  • Hemolysis: Any condition characterized by a significantly decreased erythrocyte life span.
  • Compensated hemolytic state: A state of hemolysis in which the resulting increased erythrocyte production is able to keep up with accelerated rbc destruction, thus averting any anemia.
  • Hemolytic anemia: A state of hemolysis in which increased erythrocyte production is insufficient to keep up with accelerated rbc destruction, thus producing anemia. This anemia is characterized as normochromic/normocytic, except when sufficient outpouring of the larger reticulocytes produces a resulting elevation of the MCV.

II. Diagnosis of hemolytic anemia

Diagnosis of hemolytic anemia is performed in four steps:

1.     Establish that anemia exists.

The diagnosis of anemia has been previously covered.

2.     Look for marrow response

The sine qua non for the diagnosis of hemolysis is demonstration of an attempted marrow response to erythrocyte destruction. The classic way to do this is with the reticulocyte count. Remember that you must correct the count for the degree of anemia to prevent overdiagnosis of hemolysis. The absolute retic count (in cells/µL) or, better, the reticulocyte production index (RPI) can be used to avoid this pitfall. Even so, one should never take a positive result out of context . A classic cause of reticulosis is recovery from a nutritional anemia (esp. iron and folate). For this reason, you also need corroborating evidence of erythrocyte destruction, thus:

3.     Look for erythrocyte detritus

We have previously discussed the fate of destroyed red cells and their component catabolites, such as free hemoglobin, methemoglobin, methemalbumin, bilirubin, and urobilinogen, as well as the specific binding proteins for these catabolites, such as haptoglobin and hemopexin. Laboratory measurement of some or all of these assists in the diagnosis of hemolysis.

4.     Establish the pathophysiological mechanism of hemolysis

The first distinction to make is to determine whether the hemolysis is taking place in the sinusoids of the reticuloendothelial system (extravascular hemolysis) or in the bloodstream proper (intravascular hemolysis). Both types produce indirect hyperbilirubinemia, urobilinogen in stool and urine, decreased serum haptoglobin, and reticulocytosis. In addition, assuming hemolysis is brisk enough to overwhelm the haptoglobin hemoglobin salvage mechanism, intravascular hemolysis produces hemosiderin in the urine sediment, free hemoglobin in the serum (which may be grossly visible), and free denatured hemoglobin in the urine. Some intravascular hemolytic conditions due to mechanical destruction of rbc's produce the helmet-shaped schizocytes (or "schistocytes"), which can be seen on the routine peripheral blood film. Extravascular hemolytic anemias may produce spherocytes, which are the result of an rbc having a narrow escape from the clutches of the RES.

The next determination to make is the mechanism of rbc destruction. Performing a thorough history and physical (including family and drug history), examining the peripheral blood film, and ordering a few inexpensive laboratory tests, such as the direct antiglobulin (Coombs') test for autoantibodies directed against the rbc membrane antigens and the hemoglobin electrophoresis, will lead you into Diagnosisland in 95+% of the cases. Rare cases will require labor-intensive, costly tests that have to be sent away somewhere like King of Prussia, Pennsylvania, or that are batched for six months in a dusty, coffee-stained research lab tucked away in a closet into which the Medicare inspector has yet to stumble.

III. Specific Conditions

Let us consider selected hemolytic anemias individually. These particular diseases are covered either because they are common or because they illustrate important pathophysiologic features (or both).

A. Mechanical hemolytic anemias

These are certainly the easiest to understand, even to the most concrete of thinkers. Red cells are destroyed due to hydrodynamic turbulence when they are forced over gross obstructions (such as an artificial heart valve) or "clotheslined" by innumerable fibrin strands in such microangiopathic conditions as disseminated intravascular coagulation (better known by the machonym "DIC," covered later in the heme bloc) or thrombotic thrombocytopenic purpura (TTP), an uncommon and mysterious disease of unknown etiology. The hallmark of microangiopathic hemolytic anemia is the presence of schizocytes on the routine blood film.



Schizocytes photo

B. Immunohemolytic anemias

In autoimmune hemolytic anemias, the body discourteously mounts an immune attack against its own rbc membrane antigens. This condition not surprisingly tends to occur in states characterized by systemic autoimmunity, such as lupus erythematosus. If the autoantibody is of the IgG class, hemolysis will usually occur at any temperature ( "warm autoimmune hemolytic anemia" ). Several drugs are known to produce warm autoimmune hemolytic anemia which goes away after withdrawal of the drug. Typically the antibody in warm hemolysis is one directed against a universal component of the Rh system absent only in individuals (usually of native Australian blood) with the extremely rare Rh-null rbc membrane phenotype

Autoantibodies of the IgM class typically produce cold agglutinin syndrome, in which the patient is at greater risk of symptoms in a low-temperature environment. Cold agglutinin syndrome may occasionally occur transiently in cases of Mycoplasma pneumonia and rarely infectious mononucleosis. Most cold autoagglutinins are directed against the I antigen, found in almost all adults. The rare infectious mono cold agglutinin has been characterized as anti-i.

Paroxysmal cold hemoglobinuria is a very rare syndrome in which intravascular hemolysis is produced upon exposure to cold temperature by an IgG autoantibody directed against the P antigen found on the red cells of nearly all individuals.

In alloimmune hemolytic anemia, the body synthesizes antibodies against red cell antigens foreign to the host. These antibodies may be naturally occurring (such as those directed against ABO blood group antigens) or acquired as a result of blood transfusion (including that from a fetus to its pregnant mother). Acquired antibodies include those directed against the Rh, Kell, Duffy, and Kidd system antigens. Clinical hemolysis occurs 1) when maternal antibodies send a raiding party across the placenta to raise a little hell in Fetusville (to produce hemolytic disease of the newborn „ "erythroblastosis fetalis"), and 2) when host antibodies destroy transfused red blood cells in a hemolytic transfusion reaction (which fortunately is very rare with modern blood banking practices).

Diagnosis of immunohemolytic anemia is made by demonstrating (after having proved hemolysis is occurring, as discussed above) a positive result on a simple agglutination test to demonstrate that antibodies are present on the surface of the patient's rbc's. This test is properly called the direct antiglobulin test. All but the most pedantic eschew this term, preferring the eponymous designation direct Coombs' test. Another term for immunohemolytic anemia is, therefore, "Coombs'-positive hemolytic anemia." The diagram below illustrates the principle of the Coombs' test:

Diagram: direct Coombs' test

A blood sample from the patient has the plasma poured off and the cells washed. The rbc's are resuspended in an aqueous medium. Antiserum containing antibodies against human immunoglobulin (prepared by diabolically injecting cute, innocent little animals with doses of human immunoglobulin) is then added to the patient's cells. A positive result is indicated grossly (or occasionally microscopically) by a visible agglutination reaction.

Treatment of immunohemolytic anemias is aimed at reducing the activity of the body's misdirected immune system. Glucocorticoids are the mainstay of therapy, although refractory cases can be treated with other immunosuppressive drugs and by splenectomy. This latter may be of benefit since immunohemolytic anemias most commonly are due to extravascular destruction of the auto-opsonized cells, at least some of which may occur in the spleen. Transfusion is of limited or no benefit (unless you can find a few Australian aboriginal blood donors), since the patient's autoantibodies very willingly go to work on the transfused red cells with as much relish as they do on the patient's own.

Myelodysplastic syndromes (MDS)

         The myelodysplastic syndromes (MDS, formerly known as "preleukemia") are a diverse collection of hematological (blood-related) medical conditions that involve ineffective production (or dysplasia) of the myeloid class of blood cells.

Patients with MDS often develop severe anemia and require frequent blood transfusions. In most cases, the disease worsens and the patient develops cytopenias (low blood counts) due to progressive bone marrow failure. In about one third of patients with MDS, the disease transforms into acute myelogenous leukemia (AML), usually within months to a few years.

The myelodysplastic syndromes are all disorders of the stem cell in the bone marrow. In MDS, hematopoiesis (blood production) is disorderly and ineffective. The number and quality of blood-forming cells decline irreversibly, further impairing blood production.


French-American-British (FAB) classification

In 1974 and 1975, a group of pathologists from France, the US, and Britain produced the first widely used classification of these diseases. This French-American-British classificationwas published in 197 and revised in 1982. Cases were classified into five categories: (ICD-O codes are provided where available)





Refractory anemia (RA)

characterized by less than 5% primitive blood cells (myeloblasts) in the bone marrow and pathological abnormalities primarily seen in red cell precursors


Refractory anemia with ringed sideroblasts (RARS)

also characterized by less than 5% myeloblasts in the bone marrow, but distinguished by the presence of 15% or greater red cell precursors in the marrow being abnormal iron-stuffed cells called "ringed sideroblasts"


Refractory anemia with excess blasts (RAEB)

characterized by 5-20% myeloblasts in the marrow


Refractory anemia with excess blasts in transformation (RAEB-T)

characterized by 21-30% myeloblasts in the marrow (>30% blasts is defined as acute myeloid leukemia)


Chronic myelomonocytic leukemia (CMML), not to be confused with chronic myelogenous leukemia or CML

characterized by less than 20% myeloblasts in the bone marrow and greater than 1000 * 109/uL monocytes (a type of white blood cell) circulating in the peripheral blood.

A table comparing these is available from the Cleveland Clinic

The best prognosis is seen with refractory anemia with ringed sideroblasts and refractory anemia, where some non-transplant patients live more than a decade (the average is on the order of three to five years, although long-term remission is possible if a bone marrow transplant is successful). The worst outlook is with RAEB-T, where the mean life expectancy is less than 1 year. About one quarter of patients develop overt leukemia. The others die of complications of low blood count or unrelated disease. The International Prognostic Scoring System is another tool for determining the prognosis of MDS, published in Blood in 1997. This system takes into account the percentage of blasts in the marrow, cytogenetics, and number of cytopenias.

The FAB classification was used by pathologists and clinicians for almost 20 years.

World Health Organization

In the late 1990s a group of pathologists and clinicians working under the World Health Organization (WHO) modified this classification, introducing several new disease categories and eliminating others. Most recently the WHO has evolved a new classification scheme (2008) which is based more on genetic findings. However, morphology of the cells in the peripheral blood, bone marrow aspirate, and bone marrow biopsy is still the screening test used in order to decide which classification is best and which cytogenetic aberrations may be related.

The list of dysplastic syndromes under the new WHO system includes:

Old system

New system

Refractory anemia (RA)

Refractory cytopenia with unilineage dysplasia (Refractory anemia, Refractory neutropenia, and Refractory thrombocytopenia)

Refractory anemia with ringed sideroblasts (RARS)

Refractory anemia with ring sideroblasts (RARS)

Refractory anemia with ring sideroblasts - thrombocytosis (RARS-t) (provisional entity) which is in essence a 
myelodysplastic/myeloproliferative disorder and usually has a JAK2 mutation (janus kinase) - New WHO classification 2008


Refractory cytopenia with multilineage dysplasia (RCMD) includes the subset Refractory cytopenia with multilineage dysplasia and ring sideroblasts (RCMD-RS). RCMD includes patients with pathological changes not restricted to red cells (i.e., prominent white cell precursor and platelet precursor (megakaryocyte) dysplasia.

Refractory anemia with excess blasts (RAEB)

Refractory anemia with excess blasts I and II. RAEB was divided into *RAEB-I (5-9% blasts) and RAEB-II (10-19%) blasts, which has a poorer prognosis than RAEB-I. Auer rods may be seen in RAEB-II which may be difficult to distinguish from acute myeloid leukemia.

Refractory anemia with excess blasts in transformation (RAEB-T)

The category of RAEB-T was eliminated; such patients are now considered to have acute leukemia. 5q- syndrome, typically seen in older women with normal or high platelet counts and isolated deletions of the long arm of chromosome 5 in bone marrow cells, was added to the classification.

Chronic myelomonocytic leukemia (CMML)

CMML was removed from the myelodysplastic syndromes and put in a new category of myelodysplastic-myeloproliferative overlap syndromes.


5q- syndrome


Myelodysplasia unclassifiable (seen in those cases of megakaryocyte dysplasia with fibrosis and others)


Refractory cytopenia of childhood (dysplasia in childhood) - New WHO classification 2008

Not all physicians concur with this reclassification. This is because the underlying pathology of the diseases is not well understood. It is difficult to classify things that are not well understood.

The median age at diagnosis of a MDS is between 60 and 75 years; a few patients are younger than 50; MDS diagnoses are rare in children. Males are slightly more commonly affected than females. Signs and symptoms are nonspecific and generally related to the blood cytopenias:

§                    Anemia—chronic tiredness, shortness of breath, chilled sensation, sometimes chest pain

§                    Neutropenia (low neutrophil count) —increased susceptibility to infection

§                    Thrombocytopenia (low platelet count) —increased susceptibility to bleeding and ecchymosis (bruising), as well as subcutaneoushemorrhaging resulting in purpura or petechia

Many individuals are asymptomatic, and blood cytopenia or other problems are identified as a part of a routine blood count:

§                    neutropeniaanemia and thrombocytopenia (low cell counts of white and red blood cells, and platelets, respectively);

§                    splenomegaly or rarely hepatomegaly;

§                    abnormal granules in cells, abnormal nuclear shape and size; and/or

§                    chromosomal abnormalities, including chromosomal translocations and abnormal chromosome number.

MDS must be differentiated from anemiathrombocytopenia, and/or leukopenia. Usually, the elimination of other causes of these cytopenias, along with a dysplastic bone marrow, is required to diagnose a myelodysplastic syndrome.

Blood smear from an adult female with a myelodysplastic syndrome related to radiotherapy and chemotherapy for Hodgkin disease. A hypogranular neutrophil with a pseudo-Pelger-Huet nucleus is shown. The red blood cells show marked poikilocytosis, in part related to post-splenectomy status. (Wright-Giemsa stain).

A typical investigation includes:

§                    Full blood count and examination of blood film. The blood film morphology can provide clues about hemolytic anemia, clumping of theplatelets leading to spurious thrombocytopenia, or leukemia.

§                    Blood tests to eliminate other common causes of cytopenias, such as lupushepatitisB12folate, or other vitamin deficiencies, renal failure or heart failureHIVhemolytic anemiamonoclonal gammopathy. Age-appropriate cancer screening should be considered for allanemic patients.

§                    Bone marrow examination by a hematopathologist. This is required to establish the diagnosis, since all hematopathologists consider dysplastic marrow the key feature of myelodysplasia.

§                    Cytogenetics or chromosomal studies. This is ideally performed on the bone marrow aspirate. Conventional cytogenetics requires a fresh specimen, since live cells are induced to enter metaphase to enhance chromosomal staining. Alternatively, virtual karyotyping can be done for MDS, which uses computational tools to construct the karyogram from disrupted DNA. Virtual karyotyping does not require cell culture and has dramatically higher resolution than conventional cytogenetics, but cannot detect balanced translocations.

§                    Flow cytometry is helpful to establish the presence of any lymphoproliferative disorder in the marrow.

Anemia dominates the early course. Most symptomatic patients complain of the gradual onset of fatigue and weakness, dyspnea, and pallor, but at least half the patients are asymptomatic and their MDS is discovered only incidentally on routine blood counts. Previous chemotherapy or radiation exposure is an important historic fact. Fever and weight loss should point to a myeloproliferative rather than myelodysplastic process. Children with Down syndrome are susceptible to MDS, and a family history may indicate a hereditary form of sideroblastic anemia or Fanconi anemia.

The average age at diagnosis for MDS is about 65 years, but pediatric cases have been reported. Some patients have a history of exposure to chemotherapy (especially alkylating agents such as melphalancyclophosphamidebusulfan, and chlorambucil) or radiation (therapeutic or accidental), or both (e.g., at the time of stem cell transplantation for another disease). Workers in some industries with heavy exposure to hydrocarbons such as the petroleum industry have a slightly higher risk of contracting the disease than the general population. Males are slightly more frequently affected than females. Xylene and benzene exposure has been associated with myelodysplasia. Vietnam veterans that were exposed to Agent Orange are at risk of developing MDS.

The features generally used to define a MDS are: blood cytopenias; ineffective hematopoiesis; dyserythropoiesis; dysgranulopoiesis; dysmegakaropoiesis and increased myeloblast.

Dysplasia can affect all three lineages seen in the bone marrow. The best way to diagnose dysplasia is by morphology and special stains (PAS) used on the bone marrow aspirate and peripheral blood smear. Dysplasia in the myeloid series is defined by:

§                    Granulocytic series

1.              Hypersegmented neutrophils (also seen in Vit B12/Folate deficiency)

2.              Hyposegmented neutrophils (Pseudo-Pelger Huet)

3.              Hypogranular neutrophils or pseudo Chediak Higashi large granules

4.              Auer rods - automatically RAEB II (if blast count <5% in the peripheral blood and <10% in the bone marrow aspirate) also note Auer rods may be seen in mature neutrophils in AML with translocation t(8;21)

5.              Dimorphic granules (basophilic and eosinophilic granules) within eosinophils

§                    Erythroid series

1.              Binucleated erythroid percursors and karyorrhexis

2.              Erythroid nuclear budding

3.              Erythroid nuclear strings or internuclear bridging (also seen in congenital dyserythropoietic anemias)

4.              Loss of E-cadherin in normoblasts is a sign of aberrancy

5.              PAS (globular in vacuoles or diffuse cytoplasmic staining) within erythroid precursors in the bone marrow aspirate (has no bearing on paraffin fixed bone marrow biopsy). Note: One can see PAS vacuolar positivity in L1 and L2 blasts (AFB classification; the L1 and L2 nomenclature is not used in the WHO classification)

6.              Ringed sideroblasts seen on Prussian blue iron stain (10 or more iron granules encircling 1/3 or more of the nucleus and >15% ringed sideroblasts when counted amongst red cell precursors)

§                    Megakaryocytic series (can be the most subjective)

1.              Hyposegmented nuclear features in platelet producing megakaryocytes (lack of lobation)

2.              Hypersegmented (osteoclastic appearing) megakaryocytes

3.              Ballooning of the platelets (seen with interference contrast microscopy)




In its broadest sense, anemia is a functional inability of the blood to supply the tissue with adequate O2 for proper metabolic function. Anemia is not a disease, but rather the expression of an underlying disorder or disease. Anemia is usually associated with decreased levels of hemoglobin and/or a decreased packed cell volume (hematocrit), and/or a decreased RBC count.

 Classification:  There are two main ways to classify the anemias:


     a.  Morphologicall (based on red cell size and hemoglobin content)


(1)     Microcytic hypochromic

 (MCV < 80 mcm 3, diameter of erythrocyte < 6,5 mcm)

(2)     Macrocytic hyperchromic

 (MCV - mean corpuscular volume >100 mcm3, diameter of erythrocyte > 8 mcm)

(3)     Normocytic normochromic

(MCV = 81-99 mcm 3, diameter of erythrocyte  = 7,2-7,5 mcm)

     b.  Pathophysiological (based on causative factors)


         (1)  Blood Loss

         (2)  Impaired RBC production

         (3)  Increased RBC destruction


Pathophysiological Classification of Anemias

  I.  Blood Loss Anemia

      A.  Acute posthemorrhagic anemia

      B.  Chronic posthemorrhagic anemia

 II.  Impaired RBC Production

      A.  Disturbance of the proliferation and differentiation of stem cells

          1.  Multipotential stem cells

              a.  Aplastic anemia

          2.  Unipotential stem cells

              a.  Pure red cell aplasia

              b.  Anemia of renal failure

              c.  Anemia of endocrine disease

      B.  Disturbance of the proliferation and maturation of differentiated stem cells

          1.  Defective DNA synthesis (megaloblastic anemia):

              a.  Vitamin B12 deficiency

              b.  Folic acid deficiency

          2.  Defective hemoglobin synthesis (hypochromic  anemias): 

              a.  Iron deficiency anemia (defective heme synthesis)

              b.  Thalassemia (defective globin synthesis)

          3.  Unknown or multiple mechanisms

              a.  Anemia associated with bone marrow infiltration

              b.  Anemia of chronic disease

              c.  Lead poisoning

              d.  Sideroblastic anemia

III.  Increased RBC Destruction (Hemolytic Anemias)

      A.  Intrinsic Disorders

          1.  Membrane Defects

              a.  Hereditary spherocytosis

              b.  Hereditary ovalocytosis

              c.  Hereditary acanthocytosis (rare)

              d.  Hereditary stomatocytosis (rare)

              e.  Paroxysmal nocturnal hemoglobinuria (PNH)

          2.  Metabolic Defects

              a.  Glucose-6-phosphate dehydrogenase deficiency

              b.  Pyruvate kinase deficiency

         3.  Hemoglobin Defects

              a.  Hemoglobinopathies

              b.  Thalassemia

      B.  Extrinsic Disorders

          1.  Chemical Agents

          2.  Vegetable and Animal Poisons

          3.  Infectious Agents

          4.  Physical Agents

              a.  Heat

              b.  Traumatic hemolysis (Red Cell Fragmentation Syndrome)

          5.  Immunologically Caused Hemolytic Anemias



Laboratory Investigation of Anemia


Blood count ‑        red blood cell count



A.  Morphology of red blood cell may yield important clues

    1. Size

    2. Chromia, or color

    3. Poikilocytosis ‑ abnormally shaped cells due to either premature aging or abnormal maturation.

        a. Crenated cell ‑ artifact of slide preparation.  From use of a hypertonic staining fluid ‑draws water out of the cell.

        b. Burr cell ‑ cell has blunt horns seen in anemias associated with chromic kidney diseases i.e. uremia

        c.  Acanthocytes ‑ due to error of lipoprotein metabolism/rare.  Associated with mental retardation.

        d.  Elliptocytosis ‑ cells oval in shape.  May be a genetic defect.  Homozygous – causes hemolytic anemia. May also be seen in cases of severe burns

        e.  Sickle cells ‑   contain abnormal Hgb.  Upon oxygen deprivation, the cells form a sickle shape.

        f.  Howell ‑ Jolly bodies ‑ remnant of DNA or RNA. Commonly seen in splenectomy patients.

        g.  Cabot rings ‑    similar to Howell Jolly bodies

        h.  Basophilic stippling ‑  due to poisoning by heavy metals, especially lead. Results from precipitation of ribosomal material within the red cell.

        i.  Pappenheimer bodies ‑   also called Siderocytes.  Can get them in small numbers in all anemias except Fe deficiency. Must be specially stained.  Maturation defect.

        j.  Schistocytes  irregularly shaped fragments of red cells which may be seen in hemolytic anemias.

Polychromasia ‑ (Reticulocytosis)

Polychromatophilic erythrocytes are newly released red cells, lilac in color and contain residual RNA which can be stained with a supravital stain such as New Methylene Blue. Levels of 0. 5‑1.5 % are usually considered normal.  A lot of polychromasia indicates increased cell production, usually hemolysis.  Lots of new cells have to be produced to keep up with the loss.

Blood Loss Anemia

Posthemorrhagic anemia is an anemia which develops as a result of hemorrhage. There are two types of anemias of this group according to the character of hemorrhage: 1) acute posthemorrhagic and 2) chronic posthemorrhagic anemia.

Acute posthemorrhagic anemia arises after fast massive hemorrhage as a result wounding of vessels or their damage by pathological process.

Chronic posthemorrhagic anemia develops after repeated hemorrhages, caused by injury of blood vessels during number  of diseases (dysmenorrhea, ulcer of  stomach, hemorrhoids etc.)


The picture of blood of acute posthemorrhagic anemias depends on time which has passed after hemorrhage. Depending on it it is possible pick out three periods, each of them is characterized by the certain picture of peripheral blood.

1. The first several hours after acute hemorrhage. At this period of time the total amount of blood, and also total number of erythrocytes in an organism decreases. However in unit of blood volume   the contents of erythrocytes and concentration of hemoglobin is normal.

2. The period of time from several hours untill several days after acute hemorrhage. Dillution of blood takes place as a result of transition of liquid from interstitial spaces into blood vessels. As a result of it the quantity of erythrocytes and hemoglobin in unit of volume of blood decreases, as well as hematocrit. A color index stays without changes (normochromic anemia). Qualitative changes of erythrocytes in  blood smear are not found yet.

3. The period of time from several days untill 1-2 weeks after acute hemorrhage. The most typical feature of  picture of blood in this period is occurrence of  plenty regenerative forms of erythrocytes, due to amplification of erythropoiesis in  red bone marrow. Because young unripe erythrocytes contain less hemoglobin in comparison with mature cells, the color index decreases also and anemia becomes hypochromic.


During chronic posthemorrhagic anemia after the loss of iron hematologic attributes of irondeficiency anemia develop: concentration of hemoglobin and color index decrease, in blood smear there are degenerate forms of erythrocytes (micro- and poikilocytosis, hypochromy). Quantity of erythrocytes and hematocrit may remain without changes.

Iron-deficiency anemia


Iron has a pivotal role in many metabolic processes, and the average adult contains 3–5 g of iron, of which two-thirds is in the oxygen carrying molecule haemoglobin.

A normal Western diet provides about 15·mg of iron daily, of which 5–10% is absorbed (~·1·mg), principally in the duodenum and upper jejunum, where the acidic conditions help the absorption of iron in the ferrous form. Absorption is helped by the presence of other reducing substances, such as hydrochloric acid and ascorbic acid. The body has the capacity to increase its iron absorption in the face of increased demand, for example, in pregnancy, lactation, growth spurtsand iron deficiency. Once absorbed from the bowel, iron is transported across the mucosal cell to the blood, where it is carried by the protein transferrin to developing red cells in the bone marrow. Iron stores comprise ferritin, a labile and readily accessible source of iron and haemosiderin, an insoluble form found predominantly in macrophages. About 1·mg of iron a day is shed from the body in urine, faeces, sweat and cells shed from the skin and gastrointestinal tract. Menstrual losses of an additional 20·mg per month and the increased requirements of pregnancy (500–1000·mg) contribute to the higher incidence of iron deficiency in women of reproductive age.


Causes of iron deficiency anaemia


Reproductive system

• Menorrhagia

Gastrointestinal tract


• Oesophagitis

• Oesophageal varices

• Hiatus hernia (ulcerated)

• Peptic ulcer

• Inflammatory bowel disease

• Haemorrhoids (rarely)

• Carcinoma: stomach, colorectal

• Angiodysplasia

• Hereditary haemorrhagic telangiectasia (rare)


• Coeliac disease

• Atrophic gastritis (also may result from iron deficiency)


• Growth spurts (especially in premature infants)

• Pregnancy


• Vegans

• Elderly

• Other

• Patients with chronic renal failure undergoing haemodialysis and receiving erythropoietin

Laboratory investigation of iron-deficiency anemia


1.    Total blood count.




RBC parameters:

·  MCV (mean corpuscular volume – N 75-95mcm3)

·  MCH (mean corpuscular hemoglobin – N 24-33 picograms),

·  MCHC (mean corpuscular hemoglobin concentration – N 30-38 %),

·  RDW (red cell distribution width N 11,5-14 %).

At the onset of disease amount of red blood cells is not reduced, but they are reduced in size (microcytes) and not saturated enough with hemoglobin (hypochromiya). Color index is low  (0,7-0,5) and MCHC also decreases. In blood smears small hypochromic erythrocytes are dominated with unequal size and shape (anizocytosis, poykilocytosis). In severe anemia erythroblasts may appear. The number of reticulocytes does not change. But if the anemia is caused by acute hemorrhage, immediately after it, the level of reticulocytes is increased, which is an important sign of bleeding. Osmotic resistance of erythrocytes is not changed or is slightly increased. The level of platelets does not change, only in case of hemorrhage is slightly increased.

2. Biochemical profile.

Ferritin, serum

Ferritin is a high molecular weight protein that consists of approximately 20 % iron. It is found in all cells, but especially in hepatocytes and reticuloendothelial cells, where it serves as an iron reserve. A small amount is present in plasma and serum and reflects iron stores in normal individuals. Iron is released from ferritin and binds to transferrin for transport to developing red blood cells in the bone marrow. Inadequate iron stores may result in iron deficient erythropoiesis.

Ferritin is also a useful screening test to distinguish iron deficiency from thalassemia minor in patients with anemia and erythrocyte microcytosis; ferritin is decreased in iron deficiency and normal or increased in thalassemia.


Reference Range: 45-340 ng/dL

· Values less than 100 ng/dL usually indicate depleted iron stores

· Ferritin is decreased in patients with an iron-depletion state

· Ferritin may be normal to mildly increased in functional iron deficiency

(anemia of inflammation) and in anemia secondary to thalassemia minor

· Ferritin is increased in inflammation

· Ferritin may be markedly increased in hereditary hemochromatosis and

other iron overload states, acute hepatitis, and many malignancies and in Gaucher’s disease

Iron, serum

Ingested iron is absorbed primarily from the intestinal tract, temporarily stored as ferritin in mucosal cells, and then released into the blood as Fe3+ - transferrin in equilibrium with a very small amount of free Fe3+. Serum iron can be used as one test to evaluate patients for iron deficiency, especially in combination with iron binding capacity (transferrin and transferrin saturation). Serum iron alone is unreliable due to considerable physiologic variation in the results.

Many normal subjects demonstrate a predictable diurnal variation with highest values in the morning and lowest values in the evening. Values in an individual may vary 10-40 % within a single day or day-to-day due to changes in iron absorption, marrow iron uptake, or storage iron outflow. Therefore, serum iron results should always be interpreted in the context of other studies.


Reference Range: 50-150 ug/dL

· Useful for diagnosis of iron depletion states especially when used in combination with transferrin and transferrin saturation

· Can be used for evaluation of chronic iron overload states

· Subject to physiologic variation including diurnal variation, and variation in response to iron therapy

Iron binding capacity, total (total serum transferrin) N- 1,7-4,7 mg/l, or 30,6 -84,6 micromole/l

Soluble transferrin receptor (sTfR), serum

Transferrin receptors are present on the external surface of the plasma membrane. In order for iron to be internalized into cells, the iron-transferrin complex binds to these receptors. It is then internalized through endosomes and the iron released into the cytoplasm. Proteolytic cleavage of the transferrin receptor releases a truncated version of the transferrin receptor as soluble transferrin receptor circulating in the blood. Membrane expression of transferrin receptors (TfR) are regulated by iron status. There is increased expression of TfR in iron deficiency states and this results in an increase in soluble TfR as well. In iron repletion states, there is a decrease in membrane and soluble TfR. TfR is not an acute phase reactant. While ferritin, which is an acute phase reactant, increases in response to inflammatory states, malignancy, infection, and chronic disease, soluble TfR is not affected by these confounding pathologies and may help determine the status of iron stores in patients with inflammation. TfR should not be used routinely for evaluation of iron status as it is referral test for most hospital laboratories with a higher cost and slower turn around time than ferritin, transferrin and transferrin saturation.


Reference Range: 1.8 – 4.6 mg/L

· Useful for evaluating iron status in patients with inflammation

· sTfR concentration is inversely related to iron status; sTfR elevates in

response to iron deficiency and decreases in response to iron repletion

· Patients with hemolysis or recent blood loss may have falsely elevated

sTfR levels

· sTfR is elevated in patients with thalassemia and sickle cell disease.

Transferrin, serum

Transferrin is the principle plasma protein for transport of iron. Its concentration

correlates with the total iron-binding capacity of serum. For diagnosis of iron depletion states, transferrin and iron-binding capacity may be used interchangeably. Transferrin is synthesized primarily in the liver. In otherwise healthy individuals with iron depletion states, transferrin levels in serum increase due to an increase in synthesis. High levels can be seen in pregnancy and during estrogen administration. Decreased transferrin may be seen in chronic liver disease, malnutrition, and protein loss. It is important to note that transferrin is decreased in malignancy and in both acute and chronic inflammation.


Reference Range: 200-400 mg/dL

· Transferrin is significantly increased in iron depletion states

· Transferrin is decreased in inflammatory states including anemia of chronic inflammation (functional iron deficiency)

· May be decreased in malnutrition, chronic liver disease, malignancy, and protein loss

· High levels of transferrin may be seen in pregnancy (and may be an indicator of iron depletion) and during estrogen administration.

Transferrin Saturation

Transferrin, the principal plasma protein for transport of iron, binds iron strongly at

physiologic pH. Transferrin is generally 20-45% saturated with iron. The additional amount of iron that can be bound is the unsaturated iron-binding capacity (UIBC). The sum of the serum iron and UBIC represents the total iron-binding capacity (TIBC). TIBC is an indirect measure of transferrin concentration and the two terms are often used interchangeably. The transferrin saturation (TSat) is usually reported as percent saturation (100 x serum iron/TIBC or transferrin).


Reference Range: 20-45 %

·Transferrin saturation less than 20 % is indicative of an iron deficiency state, either, latent iron deficiency, functional iron deficiency (usually associated with a decrease in transferrin but a disproportionately larger decrease in iron resulting in a transferrin saturation < 20 %) or true iron deficiency where a decrease in serum iron is associated with an increase in transferrin

· Transferrin saturation > 45 % may suggest hereditary hemochromatosis

· Transferrin saturation > 45 % may indicate an iron overload state (hemosiderosis) due to multiple transfusions or iron poisoning

· A transient increase in transferrin saturation is seen after intravenous iron infusion. The duration is dependent on the type and dose of the iron infusion.

3. Investigation of bone marrow.

Diagnostic bone marrow sampling is seldom performed in simple iron defi ciency, but, if the diagnosis is in doubt, a marrow aspirate may be carried out to demonstrate absent bone marrow stores.

4. Investigation of free protoporphyrin in erythrocytes.

Porphyrins are widely distributed in living cells throughout nature and play essential roles in various metabolic processes such as photosynthesis, transportation of oxygen and cellular respiration. In man the most important of the porphyrins is protoporphyrin 9, type III, which in combination with iron and specific proteins forms such compounds as hemoglobin, myoglobin, cytochrome,

peroxidase and catalase. In addition to its occurrence in these compounds, protoporphyrin has been found in apparently uncombined or free form in erythrocytes, hence the term "free erythrocyte protoporphyrin"


Adults: 16-36 mcg/dL red cells (0.28-0.64 micromol/L red cells)

Hemolytic anemias

Red cells being enucleated, their survival within the blood circulation depends on the stability of their various components that cannot be replaced through protein synthesis. The normal life span of the red cell is 120 days. The normal red blood cell production compensates for the destruction and loss of red cells. Thus one in every 120 red cells (or 0.8%) is replaced every day by a newly formed red cell. These newly formed red cells, called "reticulocytes", can be recognized with

special stains because of the transient persistence of ribosomes in their cytoplasm.

Hemolysis is defined as a shortening of the red cell survival below the normal of 120 days.



1. Fragmentation:

Mechanical destruction of the red cells. Examples include red cells trapped by malfunctioning artificial heart valves or fragmentation of red cells that are going through fibrin clots in the microcirculation (e.g. disseminated intra-vascular coagulation).

2. Osmotic Lysis:

The concentration of protein in the red cell is very high (32-36 gm% of hemoglobin). This creates a high osmotic force that tends to let water and sodium "leak" into the cell. This water needs to be pumped out by a membrane pump that uses ATP as a power source. If not enough ATP can be generated (as in pyruvate kinase deficiency) then the pump does not function properly and the red cell swells with water and may ultimately explode.

3. Erythrophagocytosis:

In autoimmune hemolytic anemia, red cell coated with antibodies are taken up and destroyed by macrophages, which have receptors for the Fc portion of the antibody (immunoglobulin).

4. Complement – Mediated Hemolysis:

The "complement" system is made up of a series of serum proteins which are sequentially activated, ultimately creating an active enzyme that punches a hole in the membrane of the red cell. This results in water rushing into the red cell, thus causing osmotic lysis. The complement system may be activated by bacteria, viruses, and certain antibodies.


1.    The blood smear should be examined for potential clues about the cause of hemolysis. The reticulocyte count is elevated, indicating the increased production of red cells in an attempt to compensate for abnormal red cell losses.

2.    Biochemical changes indicate the increased turnover of red cells:

·       increased serum bilirubin (unconjugated form, or "indirect")

·       increased serum lactate dehydrogenase (L.D.H.), an enzyme released from red cells

·       increased serum iron (due to its release from the heme moiety of the hemoglobin)

 In intravascular hemolysis:

·       Decreased serum haptoglobin

·       Increased free serum hemoglobin

·       Hemosiderinuria

3.    The bone marrow is sometimes examined to clarify the diagnosis. It will then show an expansion (hyperplasia) of the erythroid precursors (decreased marrow fat and lower myeloid/erythroid ratio as the erythroid cells increase in number to compensate for the hemolysis).


Rarely, the red cell survival is directly evaluated using autologous red cells labeled with radioactive chromium-51 (51Cr).


Hemoglobinopathy: A genetic defect that results in abnormal structure of one of the globin chains of the hemoglobin molecule.

Thalassemia: A genetic defect that results in production of an abnormally low quantity of a given hemoglobin chain or chains. The defect may affect the a, b, g, or d chain, or may affect some combination of the b, g, and d chain in the same patient (but never the a and b chain together). The result is an imbalance in production of globin chains and the production of an inadequate number of red cells.

The Hb S gene is found primarily in populations of native tropical African origin (which include most African-Americans). The incidence of the gene in some African populations is as high as 40 %; in African-Americans the incidence is 8 %. Unfortunately, homozygous expression produces sickle cell disease, which is a chronic hemolytic anemia and vasoocclusive condition that usually takes the life of the patient.

Sickle cell anemia is a particularly bad disease in that not only is it a hemolytic anemia, but also a vasoocclusive condition. The clinical findings can then be divided into one of these two groups:

a. Effects of chronic hemolysis

1. Anemia. Pretty much self-explanatory

2. Jaundice, due to rapid heme turnover and subsequent generation of bilirubin

3. Cholelithiasis. It has been classically taught that sickle cell patients  are prone to the formation of calcium bilirubinate gallstones due to excess bilirubin secretion into the hepatobiliary tree.

4. Aplastic crisis. Many of us have brief episodes of marrow aplasia as a result of common viral infections. With a normal erythrocyte life span of 120 days, no anemia results from an unnoticed marrow shut-down of a few days. However, the sickle cell patients, with their markedly abbreviated RBC life span, can have a precipitous fall in hematocrit (and retic count) under such conditions. This may be life-threatening.

5. Hemolytic crisis. Most sickle cell patients establish a stable, tonic level of hemolysis. Rarely, for obscure reasons, they experience a catastrophic fall in hematocrit, increasing intensity of jaundice, and increasing reticulocyte count. This is called a “hemolytic crisis.”

b. Effects of vaso-occlusion

1. Dactylitis. Resulting presumably from infarction or ischemia of the  bones of the hands and feet, this is often the presenting manifestation of sickle cell disease in a six-months-old infant. The hands and feet are swollen and painful.

2. Autosplenectomy. In childhood, the spleen is enlarged due to excess activity in destruction of the sickled erythrocytes. Gradually, the spleen infarcts itself down to a fibrous nubbin.

3. Priapism. This refers to a painful and sustained penile erection, apparently due to sludging of sickled cells in the corpora cavernosa. Sometimes the penis has to be surgically decompressed. Repeated episodes of priapism cause the spongy erectile tissues to be replaced by fibrous tissue, with impotence being the end result.

4. Renal papillary necrosis. The physiologic function of the loops of Henle make the renal medulla an eldritch, unbodylike area of high hematocrit, high osmolarity, low pH, hemodynamic stasis, and low pO2. All of these conditions predispose to sickling and infarctive loss of the papillae of the pyramids. The result is inability to concentrate and dilute urine. Even sickle cell trait individuals may experience episodes of hematuria, presumably due to this mechanism.

5. Infarctive (painful) crisis. Increased sickling activity may be brought about by any general stress on the body, especially infection. Almost any organ may suffer acute infarction (includinmg the heart), and pain is the chief symptom.

6. Sequestration crisis. This occurs mostly in infants and young children and is characterized by sudden pooling of sickled erythrocytes in the RES and vascular compartment. This produces a sudden fall in hematocrit. Sequestration crisis may be the most common cause of death in sickle cell patients in the youngest age group.

7. Leg ulcers. After all of the disasters mentioned above, this seems trivial. However, the deep, nonhealing ulcers of skin and tela subcutanea (classically around the medial malleolus) may be the only clinical manifestation of sickle cell disease in an otherwise well-compensated patient. These may be the only bugaboo standing between the patient and a productive, financially solvent life.


Megaloblastic anemia

Megaloblastic anemia is an anemia (of macrocytic classification) that results from inhibition of DNA synthesis in red blood cell production. When DNA synthesis is impaired, the cell cycle cannot progress from the growth stage to the mitosis stage. This leads to continuing cell growth without division, which presents as macrocytosis.


1.Vitamin B12 deficiency leading to folate deficiency:

Achlorhydria-induced malabsorption

§       Deficient intake

§       Deficient intrinsic factor (pernicious anemia or gastrectomy)

§          Celiac disease

§          Biological competition for Vitamin B12 by diverticulosis, fistula, intestinal anastomosis, and infection by the marine parasiteDiphyllobothrium latum (fish tapeworm)

§          Selective Vitamin B12 malabsorption (congenital—juvenile megaloblastic anemia 1—and drug-induced)

§          Chronic pancreatitis

§          Ileal resection and bypass

§          Nitrous oxide anesthesia (usually requires repeated instances).

§          2. Folate deficiency:

§       Alcoholism

§       Deficient intake

§       Increased needs: pregnancy, infant, rapid cellular proliferation, and cirrhosis

§       Malabsorption (congenital and drug-induced)

§       Intestinal and jejunal resection

§       (indirect) Deficient thiamine and factors (e.g., enzymes) responsible for folate metabolism.

3. Combined Deficiency: Vitamin B12 & folate.

§     Inherited Pyrimidine Synthesis Disorders: Orotic aciduria

§     Inherited DNA Synthesis Disorders

a.   Toxins and Drugs:

b.   Folic acid antagonists (methotrexate)

c.    Purine synthesis antagonists (6-mercaptopurine)

d.   Pyrimidine antagonists (cytosine arabinoside)

e.    Phenytoin

f.     Nitrous Oxide


Hematological findings

The blood film can point towards vitamin deficiency:

Decreased red blood cell (RBC) count and hemoglobin levels

Increased mean corpuscular volume (MCV, >95 fl) and mean corpuscular hemoglobin (MCH)

Normal mean corpuscular hemoglobin concentration (MCHC, 32–36 g/dL)

The reticulocyte count is decreased due to destruction of fragile and abnormal megaloblastic erythroid precursor.

§     The platelet count may be reduced.

§     Neutrophil granulocytes may show multisegmented nuclei ("senile neutrophil"). This is thought to be due to decreased production and a compensatory prolonged lifespan for circulating neutrophils, which increase numbers of nuclear segments with age.

§     Anisocytosis (increased variation in RBC size) and poikilocytosis (abnormally shaped RBCs).

§     Macrocytes (larger than normal RBCs) are present.

§     Ovalocytes (oval-shaped RBCs) are present.

§     Howell-Jolly bodies (chromosomal remnant) also present.

§     Blood chemistries will also show:

§     In increased lactic acid dehydrogenase (LDH) level. The isozyme is LDH-2 which is typical of the serum and hematopoetic cells.

§     Increased homocysteine and methylmalonic acid in Vitamin B12 deficiency

§     Increased homocysteine in folate deficiency

Bone marrow  shows megaloblastic hyperplasia.


Vitamin B12 deficiency

The total amount of vitamin B12 stored in the body is between two and five mg in adults. Approximately 50 % is stored in the liver, but approximately 0.1 % is lost each day, due to secretions into the gut — not all of the vitamin in the gut is reabsorbed. While bile is the main vehicle for B12 excretion, most of the B12 secreted in bile is recycled via enterohepatic circulation.


Vitamin B12 can be found in large quantities in animal products, including meat, poultry, fish, seafood, eggs, and dairy products.


§     Inadequate dietary intake of vitamin B12. Vitamin B12occurs in animal products (eggs, meat, milk) as the animal absorbs it by grazing in soil (or consuming unwashed crops or consuming other animals or feed composed of other animals that have done so, or fortified feed, etc.); significant portions of the world population have vitamin B12deficiency, and it is theorized to be a problem of absorption as opposed to consumption. Vegan populations may have characteristic deficiency, more notably Western countries, unless one uses supplements or eats enriched food, though B12 deficiency is observed in omnivorous, vegan and vegetarian populations frequently and everyone should be tested regularly. Children are at primary risk for dietary deficiency, since they have fewer vitamin stores and a relatively larger vitamin need per calorie of intake.

§     Selective impaired absorption of vitamin B12 due to intrinsic factor deficiency. This may be caused by the loss of gastric parietal cells in chronic atrophic gastritis (in which case, the resulting megaloblastic anemia takes the name of "pernicious anemia"), or may result from wide surgical resection of stomach (for any reason), or from rare hereditary causes of impaired synthesis of intrinsic factor.

§     Impaired absorption of vitamin B12 in the setting of a more generalized malabsorption or maldigestion syndrome. This includes any form of structural damage or wide surgical resection of the terminal ileum (the principal site of vitamin B12 absorption).

§     Forms of achlorhydria (including that artificially induced by drugs such as proton pump inhibitors) can cause B12 malabsorption from foods, since acid is needed to split B12from food proteins and salivary binding proteins. This process is thought to be the most common cause of low B12 in the elderly, who often have some degree of achlorhydria without being formally low in intrinsic factor. This process does not affect absorption of small amounts of B12 in supplements such as multivitamins, since it is not bound to proteins, as is the B12 in foods.

§     Surgical removal of the small bowel (for example in Crohn's disease) such that the patient presents with short bowel syndrome and is unable to absorb vitamin B12. This can be treated with regular injections of vitamin B12.

§     Long-term use of ranitidine hydrochloride may contribute to deficiency of vitamin B12.

§     Coeliac disease may also cause impaired absorption of this vitamin, though this is due not to loss of intrinsic factor, but rather damage to the small bowel.

§     Some bariatric surgical procedures, especially those that involve removal of part of the stomach, such as Roux-en-Y gastric bypass surgery. (Procedures such as theadjustable gastric band type do not appear to affect B12 metabolism significantly).

§     Bacterial overgrowth in parts of the small bowel are thought to be able to absorb B12. An example occurs in so-called blind loop syndrome.

§     The diabetes medication metformin may interfere with B12 dietary absorption.

§     Hereditary causes such as severe MTHFR deficiencyhomocystinuria, and transcobalamin deficiency.

§     Some studies have shown that giardiasis, or similar parasitic infections may be a cause of vitamin B12 deficiency.

§     Malnutrition of alcoholism.

§     Nitrous oxide abuse.


Clinical symptoms: The main syndrome of vitamin B12 deficiency is Biermer's disease (pernicious anemia). It is characterized by a triad of symptoms:

1.   Anemia with bone marrow promegaloblastosis (megaloblastic anemia). This is due to the inhibition of DNA synthesis (specifically purines and thymidine)

2.   Gastrointestinal symptoms : These are thought to be due to defective DNA synthesis inhibiting replication in a site with a high turnover of cells. This may also be due to the autoimmune attack on the parietal cells of the stomach in pernicious anemia. There is an association with GAVE syndrome (commonly called watermelon stomach) and pernicious anemia. 

3.   Neurological symptoms: Sensory or motor deficiencies (absent reflexes, diminished vibration or soft touch sensation)subacute combined degeneration of spinal cord, or even symptoms of dementia and or other psychiatric symptoms may be present. The presence of peripheral sensory-motor symptoms or subacute combined degeneration of spinal cord strongly suggests the presence of a B12 deficiency instead of folate deficiency.

Each of those symptoms can occur either alone or along with others. The neurological complex, defined as myelosis funicularis, consists of the following symptoms:

1.   Impaired perception of deep touch, pressure and vibration, abolishment of sense of touch, very annoying and persistent paresthesias

2.   Ataxia of dorsal cord type

3.   Decrease or abolishment of deep muscle-tendon reflexes

4.   Pathological reflexes — BabinskiRossolimo and others, also severe paresis


The Schilling test is a medical investigation used for patients with vitamin B12 deficiency. The purpose of the test is to determine whether the patient has pernicious anemia.

In the first part of the test, the patient is given radiolabeled vitamin B12 to drink or eat. The most commonly used radiolabels are 57Co and 58Co. An intramuscular injection of unlabeled vitamin B12 is given an hour later. This is not enough to replete or saturate body stores of B12 (this requires about 10 B12 injections over some length of time). The purpose of the single injection is to temporarily saturate B12 receptors in the liver with enough normal vitamin B12 to prevent radioactive vitamin B12 binding in body tissues (especially in the liver), so that if absorbed from the G.I. tract, it will pass into the urine. The patient's urine is then collected over the next 24 hours to assess the absorption.

Normally, the ingested radiolabeled vitamin B12 will be absorbed into the body. Since the body already has liver receptors for transcobalamin/vitamin B12 saturated by the injection, much of the ingested vitamin B12 will be excreted in the urine.

§     A normal result shows at least 10 % of the radiolabeled vitamin B12 in the urine over the first 24 hours.

§     In patients with pernicious anemia or with deficiency due to impaired absorption, less than 10 % of the radiolabeled vitamin B12 is detected.

The normal test will result in a higher amount of the radiolabeled cobalamin in the urine because it will have been absorbed by the intestinal epithelium, but passed into the urine because all hepatic B12 receptors were occupied. An abnormal result will cause less of the labeled cobalamin to appear in the urine because it will remain in the intestine and be passed into the feces.

If an abnormality is found, i.e. the B12 in the urine is only present in low levels, the test is repeated, this time with additional oral intrinsic factor.

§     If this second urine collection is normal, this shows a lack of intrinsic factor production, or pernicious anemia.

§     A low result on the second test implies abnormal intestinal absorption (malabsorption), which could be caused by coeliac diseasebiliary disease, Whipple's disease, fish tapeworm infestation (Diphyllobothrium latum), or liver disease. Malabsorption of B12 can be caused by intestinal dysfunction from a low vitamin level in-and-of-itself, causing test result confusion if repletion has not been done for some days previously.


Myelodysplastic syndrome (MDS)


MDS are a morphologically and clinically heterogeneous group of acquired stem cell disorders characterized by ineffective and dysplastic haematopoiesis in one or more cell lines that affect predominantly older subjects. The patients usually present with anaemia and dysplastic morphologic abnormalities in cells of more than one lineage (neutropenia, thrombocytopenia, monocytosis, or a combination of types of cytopenias). The diagnosis is essentially based on the morphological examination of a blood smear and a bone marrow biopsy specimen. The French-American-British classification divides the MDS into five subtypes based on the number of immature blast cells and morphological abnormalities in blood and bone marrow and the presence of ringed sideroblasts.

The median age at diagnosis of a MDS is between 60 and 75 years; a few patients are younger than 50; MDS diagnoses are rare in children. Males are slightly more commonly affected than females. Signs and symptoms are nonspecific and generally related to the blood cytopenias:

§     Anemia (low RBC count or reduced hemoglobin) —chronic tiredness, shortness of breath, chilled sensation, sometimes chest pain

§     Neutropenia (low neutrophil count) —increased susceptibility to infection

§     Thrombocytopenia (low platelet count) —increased susceptibility to bleeding and ecchymosis (bruising), as well as subcutaneoushemorrhaging resulting in purpura or petechia

Many individuals are asymptomatic, and blood cytopenia or other problems are identified as a part of a routine blood count:

§     neutropeniaanemia and thrombocytopenia (low cell counts of white and red blood cells, and platelets, respectively);

§     splenomegaly or rarely hepatomegaly;

§     abnormal granules in cells, abnormal nuclear shape and size; and/or

§     chromosomal abnormalities, including chromosomal translocations and abnormal chromosome number.


Myeloproliferative Syndrome (Chronic Myeloproliferative Disorders, CMPD)

         The chronic myeloproliferative disorders (previously also called the myeloproliferative syndromes) include chronic myeloid leukemia (CML), osteomyelosclerosis (OMS), polycythemia vera (PV) and essential thrombocythemia (ET). Clearly, noxious agents of unknown etiology affect the progenitor cells at different stages of differentiation and trigger chronic malignant proliferation in the white cell series (CML), the red cell series (PV), and the thrombocyte series (ET). Sometimes, they lead to concomitant synthesis of fibers (OMS). Transitional forms and mixed forms exist particularly between PV, ET, and OMS.

         The chronic myeloproliferative disorders encompass chronic autonomous disorders of the bone marrow and the embryonic blood-generating organs (spleen and liver), which may involve one or several cell lines. The common attributes of these diseases are onset in middle age, development of splenomegaly, and slow disease progression (Table 20). In 95% of cases, CML shows a specific chromosome aberration (Philadelphia chromosome with a specific BCR-ABL translocation) and may make the transition into a blast crisis. PV and ET often show similar traits (high thrombocyte count or high  Hb) and have a tendency to secondary bone marrow fibrosis. OMS is primarily characterized by fibrosis in bone marrow and splenomegaly.

Hemorrhagic syndrome

Definition of the “hemorrhagic syndrome“ and “hemorrhagic diathesis.”

Bleeding from a platelet disorder is usually localized to superficial sites such as the skin and mucous membranes, comes on immediately after trauma or surgery, and is readily controlled by local measures. In contrast, bleeding from secondary hemostatic or plasma coagulation defects occurs hours or days after injury and is unaffected by local therapy. Such bleeding most often occurs in deep subcutaneous tissues, muscles, joints, or body cavities. A careful and thorough history may establish the presence of a hemostatic disorder and guide initial laboratory testing.

Hemorrhagic spots on surface of the skin, patechiae, purpura, heleangiectasis, and hemorrhages. These signs are present not only on the skin, but in subcutaneous fat, muscle, brain too. The hemorrhagic syndrome is usual in case of inherited thrombocytopenic purpuras, neonatal thrombocytopenic purpuras. The other signs of this syndrome are bleeding, hematuria, upper and lower gastrointestinal tract hemorrhage, prolonged bleeding from the umbilical stump or from veni-punctures, intracranial hemorrhage.


Platelets arise from the fragmentation of megakaryocytes, which are very large, polyploid bone marrow cells produced by the process of endomitosis. They undergo from three to five cycles of chromosomal duplication without cytoplasmic division. After leaving the marrow space, about one-third of the platelets are sequestered in the spleen, while the other two-thirds circulate for 7 to 10 days. Normally, only a small fraction of the platelet mass is consumed in the process of hemostasis, so most platelets circulate until they become senescent and are removed by phagocytic cells. The normal blood platelet count is 150,000 to 450,000/ul. A decrease in platelet count stimulates an increase in the number, size, and ploidy of megakaryocytes, releasing additional platelets into the circulation. This process is regulated by thrombopoietin (TPO) binding to its megakaryocyte receptor, a proto-oncogene c-mpl. TPO (c-mpl ligand) is secreted continuously at a low level and binds tightly to circulating platelets. A reduction in platelet count increases the level of free TPO and thereby stimulates megakaryocyte and platelet production.

The platelet count varies during the menstrual cycle, rising following ovulation and falling at the onset of menses. It is also influenced by the patient's nutritional state and can be decreased in severe iron, folic acid, or vitamin B12 deficiency. Platelets are acute-phase reactants, and patients with systemic inflammation, tumors, bleeding, and mild iron deficiency may have an increased platelet count, a benign condition called secondary or reactive thrombocytosis. The cytokines interleukin (IL)-3, IL-6, and IL-11 may stimulate platelet production in acute inflammation. In contrast, the increase in platelet count that is characteristic of the myeloproliferative disorders such as polycythemia vera, chronic myelogenous leukemia, myeloid metaplasia, and essential thrombocytosis can cause either severe bleeding or thrombosis. In these patients, unregulated platelet production is secondary to a clonal stem cell abnormality affecting all the bone marrow progenitors.



Thrombocytopenia is caused by one of three mechanisms-decreased bone marrow production, increased splenic sequestration, or accelerated destruction of platelets. In order to determine the etiology of thrombocytopenia, each patient should have a careful examination of the peripheral blood film, an assessment of marrow morphology by examination of an aspirate or biopsy, and an estimate of splenic size by bedside palpation supplemented, if necessary, by ultrasonography or computed tomographic (CT) scan. Occasional patients have "pseudothrombocytopenia," a benign condition in which platelets agglutinate or adhere to leukocytes when blood is collected with EDTA as anticoagulant. This is a laboratory artifact, and the actual platelet count in vivo is normal.

Impaired Production Disorders that injure stem cells or prevent their proliferation frequently cause thrombocytopenia. They usually affect multiple hematopoietic cell lines so that thrombocytopenia is accompanied by varying degrees of anemia and leukopenia. Diagnosis of a platelet production defect is readily established by examination of a bone marrow aspirate or biopsy, which should show a reduced number of megakaryocytes. The most common causes of decreased platelet production are marrow aplasia, fibrosis, or infiltration with malignant cells, all of which produce highly characteristic marrow abnormalities. Occasionally, thrombocytopenia is the presenting laboratory abnormality in these disorders. Cytotoxic drugs impair megakaryocyte proliferation and maturation and frequently cause thrombocytopenia. Rare marrow disorders such as congenital amegakaryocytic hypoplasia and thrombocytopenia with absent radii (TAR syndrome), produce a selective decrease in megakaryocyte production.

Splenic Sequestration Since one-third of the platelet mass is normally sequestered in the spleen, splenectomy will increase the platelet count by 30%. Postsplenectomy thrombocytosis is a benign self-limited condition that does not require specific therapy. In contrast, when the spleen enlarges, the fraction of sequestered platelets increases, lowering the platelet count. The most common causes of splenomegaly are portal hypertension secondary to liver disease and splenic infiltration with tumor cells in myeloproliferative or lymphoproliferative disorders. Isolated splenomegaly is rare, and in most patients it is accompanied by other clinical manifestations of an underlying disease. Many patients with leukemia, lymphoma, or a myeloproliferative syndrome have both marrow infiltration and splenomegaly and develop thrombocytopenia from a combination of impaired marrow production and splenic sequestration of platelets.

Accelerated Destruction Abnormal vessels, fibrin thrombi, and intravascular prostheses can all shorten platelet survival and cause nonimmunologic thrombocytopenia. Thrombocytopenia is common in patients with vasculitis, the hemolytic uremic syndrome (HUS), thrombotic thrombocytopenic purpura (TTP), or as a manifestation of disseminated intravascular coagulation (DIC). In addition, platelets coated with antibody, immune complexes, or complement are rapidly cleared by mononuclear phagocytes in the spleen or other tissues, inducing immunologic thrombocytopenia. The most common causes of immunologic thrombocytopenia are viral or bacterial infections, drugs, and a chronic autoimmune disorder referred to as idiopathic thrombocytopenic purpura (ITP). Patients with immunologic thrombocytopenia do not usually have splenomegaly and have an increased number of bone marrow megakaryocytes.



Many common drugs can cause thrombocytopenia. Cancer chemotherapeutic agents may depress megakaryocyte production. Ingestion of large quantities of alcohol has a marrow-depressing effect leading to transient thrombocytopenia, particularly in binge drinkers. Thiazide diuretics, used to treat hypertension or congestive heart failure, impair megakaryocyte production and can produce mild thrombocytopenia (50,000 to 100,000/uL), which may persist for several months after the drug is discontinued.

Most drugs induce thrombocytopenia by eliciting an immune response in which the platelet is an innocent bystander. The platelet is damaged by complement activation following the formation of drug-antibody complexes. Current laboratory tests can identify the causative agent in 10% of patients with clinical evidence of drug-induced thrombocytopenia. The best proof of a drug-induced etiology is a prompt rise in the platelet count when the suspected drug is discontinued. Patients with drug-induced platelet destruction may also have a secondary increase in megakaryocyte number without other marrow abnormalities.

Although most patients recover within 7 to 10 days and do not require therapy, occasional patients with platelet counts <10,000 to 20,000/uL have severe hemorrhage and may require temporary support with glucocorticoids, plasmapheresis, or platelet transfusions while waiting for the platelet count to rise. A patient who has recovered from drug-induced immunologic thrombocytopenia should be instructed to avoid the offending drug in the future, since only minute amounts of drug are needed to set up subsequent immune reactions. Certain drugs that are cleared from body storage depots quite slowly, such as phenytoin, may induce prolonged thrombocytopenia.

Heparin is a common cause of thrombocytopenia in hospitalized patients. Between 10 and 15% of patients receiving therapeutic doses of heparin develop thrombocytopenia and, occasionally, may have severe bleeding or intravascular platelet aggregation and paradoxical thrombosis. Heparin-induced thrombosis, sometimes called the "white clot syndrome," can be fatal unless recognized promptly. Most cases of heparin thrombocytopenia are due to drug-antibody binding to platelets; some are secondary to direct platelet agglutination by heparin. The offending antigen is a complex formed between heparin and the platelet-derived heparin neutralizing protein, platelet factor 4. Prompt cessation of heparin will reverse both thrombocytopenia and heparin-induced thrombosis. Low-molecular-weight heparin products have reduced the incidence of heparin-induced thrombocytopenia. They are effective antithrombotic agents and are less immunogenic. Unfortunately, 80 to 90% of the antibodies generated against conventional heparins cross-react with low-molecular-weight heparins, so only a minority of patients with preformed antibody can be treated with this product.



The immunologic thrombocytopenias can be classified on the basis of the pathologic mechanism, the inciting agent, or the duration of the illness. The explosive onset of severe thrombocytopenia following recovery from a viral exanthem or upper respiratory illness (acute ITP) is common in children and accounts for 90% of the pediatric cases of immunologic thrombocytopenia. Of these patients, 60% recover in 4 to 6 weeks and >90% recover within 3 to 6 months. Transient immunologic thrombocytopenia also complicates some cases of infectious mononucleosis, acute toxoplasmosis, or cytomegalovirus infection and can be part of the prodromal phase of viral hepatitis and initial infection with HIV. Acute ITP is rare in adults and accounts for <10% of postpubertal patients with immune thrombocytopenia. Acute ITP is caused by immune complexes containing viral antigens that bind to platelet Fc receptors or by antibodies produced against viral antigens that cross-react with the platelet. In addition to these viral disorders, the differential diagnosis includes atypical presentations of aplastic anemia, acute leukemias, or metastatic tumor. A bone marrow examination is essential to exclude these disorders, which can occasionally mimic acute ITP.

Most adults present with a more indolent form of thrombocytopenia that may persist for many years and is referred to as chronic ITP. Women age 20 to 40 are afflicted most commonly and outnumber men by a ratio of 3:1. They may present with an abrupt fall in platelet count and bleeding similar to patients with acute ITP. More often they have a prior history of easy bruising or menometrorrhagia. These patients have an autoimmune disorder with antibodies directed against target antigens on the glycoprotein IIb-IIIa or glycoprotein Ib-IX complex. Although most antibodies function as opsonins and accelerate platelet clearance by phagocytic cells, occasional antibodies bind to epitopes on critical regions of these glycoproteins and impair platelet function. Platelet-associated IgG can be measured but specificity is a problem. High "background" level of IgG on normal platelets and elevations in plasma immunoglobulin levels or in circulating immune complexes will nonspecifically increase platelet-associated IgG. Few clinical situations require platelet-associated IgG testing.

A low platelet count may be the initial manifestation of systemic lupus erythematosus (SLE) or the first sign of a primary hematologic disorder. Thus, patients with chronic ITP should have a bone marrow examination and an antinuclear antibody determination. In addition, patients with hepatic or splenic enlargement, lymphadenopathy, or atypical lymphocytes should have serologic studies for hepatitis viruses, cytomegalovirus, Epstein-Barr virus, toxoplasma, and HIV. HIV infection is a common cause of immunologic thrombocytopenia. Thrombocytopenia can be the initial symptom of HIV infection or a complication of fully developed clinical AIDS.

Patients with congenital plasma coagulation defects characteristically bleed into muscles, joints, and body cavities hours or days after an injury. Most of the inherited plasma coagulation disorders are due to defects in single coagulation proteins, with the two X-linked disorders, factors VIII and IX deficiency, accounting for the majority. These patients may have severe bleeding and chronic disability and require specialized medical therapy. With rare exceptions, the known disorders prolong either the prothrombin time (PT), partial thromboplastin time (PTT), or both. If they are abnormal, quantitative assays of specific coagulation proteins are then carried out using the PT or PTT tests with plasma from congenitally deficient individuals as substrate. The corrective effect of varying concentrations of patient plasma is measured and expressed as a percentage of a normal pooled plasma standard. The interval range for most coagulation factors is from 50 to 150% of this average value, and the minimal level of most individual factors needed for adequate hemostasis is 25%.

Acquired coagulation disorders are both more frequent and more complex, arising from deficiencies of multiple coagulation proteins and simultaneously affecting both primary and secondary hemostasis. The most common acquired hemorrhagic disorders are (1) disseminated intravascular coagulation (DIC), (2) the hemorrhagic diathesis of liver disease, and (3) vitamin K deficiency and complications of anticoagulant therapy.