Hemoglobin

[Physiology][Hematology] General Concepts in Hemolytic Anemias

November 18, 2016 Hematology, Physiology and Pathophysiology No comments , , , , , , , , , , , , , , , , , , , , ,

Hemolysis is the accelerated destruction of red blood cells (RBCs), leading to decreased RBC survival. The bone marrow's response to hemolysis is increased erythropoiesis, reflected by reticulocytosis. If the rate of hemolysis is modest and the bone marrow is able to completely compensate for the decreased RBC life span, the hemoglobin concentration may be normal; this is called fully compensated hemolysis. If the bone marrow is unable to completely compensate for hemolysis, the anemia occurs. This is called incompletely compensated hemolysis.

PS (from wikipedia): The Reticulocyte production index (RPI) is a calculated value used in the diagnosis of anemia. This calculation is necessary because the raw reticulocyte count is misleading in anemic patients. The problem arises because the reticulocyte count is not really a count but rather a percentage: it reports the number of reticulocytes as a percentage of the number of red blood cells. In anemia, the patient's red blood cells are depleted, creating an erroneously elevated reticulocyte count.

Calculation of RPI

Step 1 – Reticulocyte Index is calculated using the formla on the left

Step 2 – The next step is to correct for the longer life span of prematurely released reticulocytes in the blood—a phenomenon of increased red blood cell production. This relies on a table:

Hematocrit (%) Retic survival (days) = maturation correction
36-45 1.0
26-35 1.5
16-25 2.0
15 and below 2.5

Step 3 – The Reticulocyte Production Index is calcualted using the formla below:

Hemolysis can be classified as extravascular or intravascular. Extravascular hemolysis, in which erythrocyte desstruction occurs by macrophages in the liver and spleen, is more common. Intravascular hemolysis refers to RBC destruction occurring primarily within blood vessels. The distinction between intravascular and extravascular hemolysis is not absolute because both occur simultaneously, at least to some degree, in the same patient, and the manifestations of both can overlap. The site of RBC destruction in different conditions can be conceptualized to occur in a spectrum between pure intravascular and pure extravascular hemolysis. Some hemolytic anemias are predominantly intravascular, and some are predominantly extravascular. Others have substantial components of both.

To understand better, the hemolytic anemias can be classified according to whether the cause of hemolysis is intrinsic or extrinsic to the RBC.Intrinsic causes of hemolysis include abnormalities in hemoglobin structure or function, the RBC membrane, or RBC metabolism (cytosolic enzymes). Extrinsic causes may be due to a RBC-directed antibody, a disordered vasculature, or the presence of infecting organisms or toxins. In general, intrinsic causes of hemolysis are inherited and extrinsic causes are acquired, but there are notable exceptions.


Hemolysis Due to Intrinsic Abnormalities of the RBC

screen-shot-2016-11-14-at-9-19-43-pmIntrinsic causes of hemolysis include abnormalities of hemoglobin structure or function, the RBC membrane, or RBC metabolism (cytosolic enzymes). Hemoglobin is the oxygen-carrying protein within RBCs. It is composed of four globular protein subunits, called globins, each with an oxygen-binding heme group. The two main types of globins are the alpha-globins and the beta-globins, which are made in essentially equivalent amount in precursors of RBCs. Normal adult hemoglobin (Hb A) has two alpha-globins and two beta-globins (alpha2beta2).

Abnormalities of Hemoglobin

Disorders of hemoglobin can be classified as qualitative or quantitative disorders. Qualitative abnormalities of hemoglobin arise from mutations that change the amino acid sequence of the globin, thereby producing structrual and functional changes in hemoglobin. There are four ways in which hemoglobin can be qualitatively abnormal: (i) decreased solubility, (ii) instability, (iii) altered oxygen affinity, (iv) altered maintenance of the oxidation state of the heme-coordinated iron. Hemolytic anemia (qualitative abnormalities) result from decreased solubility and instability of hemoglobin. Qualitative hemoglobin disorders often are referred to as hemoglobinopathies, even though the term technicially can apply to both qualitative and quantitative disorders.

Quantitative hemoglobin disorders result from the decreased and imbalanced production of generally structurally normal globins. For example, if beta-globin production is diminished by a mutation, there will be a relative excess of alpha-globins. Such imbalanced production of alpha- and beta-globins damages RBCs and their precursors in the bone marrow. These quantitative hemoglobin disorders are called thalassemias.

Abnormalities of the RBC Membrane

Some hemolytic diseases is characterized by abnormal shape and flexibility of RBCs because of a deficiency or dysfunction of one or more of the membrane proteins, which leads to shortened RBC survival (hemolysis).

The RBC membrane consists of a phospholipid-cholesterol lipid bilayer intercalated by integral membrane proteins such as band 3 (the anion transport channel) and the glycophorins. This relatively fluid layer is stabilized by attachment to a membrane skeleton. Spectrin is the major protein of the skeleton, accounting for approximately 75% of its mass. The skeleton is organized into a hexagonal lattice. The hexagon arms are formed by fiber-like spectrin tetramers, whereas the hexagon corners are composed of small oligomers of actin that, with the aid of other proteins (4.1 and adducin), connect the spectrin tetramers into a two-dimensional lattice. The membrane cytoskeleton and its fixation to the lipid-protein bilayer are the major determinants of the shape, strength, flexibility, and survival of RBCs. When any of these constituents are altered, RBC survival may be shortened.

Abnormalities of RBC Metabolism (cytosolic enzymes)

Normal metabolism of the mature RBC involves two principal pathways of glucose catabolism: the glycolytic pathway and the hexose-monophosphate shunt. The three major functions of the products of glucose catabolism in the erythrocyte are (i) maintenance of protein integrity, cellular deformability, and RBC shape; (ii) preservation of hemoglobin iron in the ferrous form; and (iii) modulation of the oxygen affinity of hemoglobin. These functions are served by the regulation of appropriate production of five specific molecules: ATP, reduced glutathione, reduced NADH, reduced NADPH, and 2,3-BPG. Maintenance of the biochemical and structural integrity of the RBC depends on the normal function of >20 enzymes involved in these pathways as well as the availability of five essential RBC substrates: glucose, glutathione, NAD, NAD phosphate (NADP), and adenosine diphosphate (ADP).

  • ATP

The primary function of the glycolytic pathway is the generation of ATP, which is necessary for the ATPase-linked sodium-potassium and calcium membrane pumps essential for cation homeostasis and the maintenance of erythrocyte deformability.

  • 2,3-BPG

The production of 2,3-BPG is regulated by the Rapoport-Luebering shunt, which is controlled by bisphosphoglyceromutase, the enzyme that converts 1,3-BPG to 2,3-BPG. Concentration of 2,3-BPG in the RBC in the RBC in turn regulates hemoglobin oxygen affinity, thus facilitating the transfer of oxygen from hemoglobin to tissue-binding sites.

  • Reduced gluthione

The major function of the hexose-monophosphate shunt is preservation and regeneration of reduced gluthione, which protects hemoglobin and other intracellular and membrane proteins from oxidant injury.

Abnormalities of the glycolytic pathway

Defects in the glycolytic pathway lead to a decrease in the production of ATP or a change in the concentration of 2,3-BPG.

Deficiencies of erythrocyte hexokinase, glucose phosphate isomerase, phosphofructokinase, and pyruvate kinase (PK) all lead to a decrease in ATP concentration. Although genetic disorders involving nearly all of the enzymes of the glycolytic pathway have been described, PK accounts for >80% of the clinically significant hemolytic anemias from defects in this pathway. With the exception of phosphoglycerate kinase deficiency, which is X-linked, all other glycolytic enzyme defects are autosomal recessive.

PK deficiency is the most common congenital nonspherocytic hemolytic anemia caused by a defect in glycolytic RBC metabolism. The syndrome is both genetically and clinically heterogeneous. Both glucose phosphate isomerase and hexokinase deficiencies produce nonspherocytic hemolytic anemia associated with decreased ATP and 2,3-BPG content.

Abnormalities of the hexose-monophosphate shunt

G6PD deficiency is the most frequently encountered abnormality of RBC metabolism, affecting >200 million people worldwide. The gene for G6PD is carried on the X chromosome and exhibits extensive polymorphism. Enzyme deficiency is observed in males carrying a variant gene. Hemolysis in G6PD-deficient RBCs is due to a failure to generate adequate NADPH, leading to insufficient levels of reduced glutathione. This renders erythrocytes susceptible to oxidation of hemoglobin by oxidant radicals, such as hydrogen peroxide. The resulting denatured hemoglobin aggregates and forms intraerythrocytic Heinz bodies, which bind to membrane cytoskeletal proteins. Membrane proteins are also subject to oxidation, leading to decreased cellular deformability. Cells containing Heinz bodies are entrapped or partially destroyed in the spleen, resulting in loss of cell membranes through pitting of Heinz bodies and leading to hemolysis.

Abnormalities of nucleotide metabolism

Pyrimidine-5'-nucleotidase deficiency is an enzymatic abnormality of pyrimidine metabolism associated with hemolytic anemia. The peripheral blood smear in patients with this defect often shows RBCs containing coarse basophilic stippling. Lead intoxication also inactivates the enzyme, leading to an acquired variant of pyrimidine-5'-nucleotidase deficiency.

Adenosine deaminase (ADA) excess is an unusual abnormality. It is caused by a genetically determined increase in the activity of a normal erythrocyte enzyme. The excessive deaminase activity prevents normal salvage of adenosine and causes subsequent depletion of ATP and hemolysis.

Red Blood Cell Analytic Parameters

December 14, 2015 Hematology, Laboratory Medicine No comments , , , , , ,

blood_transfusionRBCs are defined by three quantitative values: the volume of packed red cells or hematocrit (Hct), the amount of hemoglobin (Hb), and the red cell concentration per unit volume. Three additional indices describing average qualitative characteristics of the red cell polupation are also collected. These are mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC).

Volume of Packed Red Cells (Hematocrit)

The hematocrit is the proportion of the volume of a blood sample that is occupied by red cells. Hct may be determined manually by centrifugation of blood at a given speed and time in a standardized glass tube with a uniform bore. The height of the column of red cells after centrifugation compared with total blood sample volume yields the Hct. However, several sources of error are inherent in the manual methods of measuring Hct technique. The spun Hct measures the red cell concentration, not red cell mass. Therefore, patients in shock or with volume depletion may have normal or high Hct measurements due to hemoconcentration despite a decreased red cell mass. In addition, technical sources of error in manual Hct determinations usually arise from inappropriate concentrations of anticoagulants, poor mixing of samples, or insufficient centrigugation. Another inherent error   in manual Hct determinations arises from trapping of plasma in the red cell column. This may account for 1% to 3% of the volume in microcapillary tube methods, with macrotube methods trapping relative more plasma. It should be noted that abnormal red cells (e.g., sickle cells, microcytic cells, macrocytic cells, or spherocytes) often trap higher volumes of plasma due to increased cellular rigidity, possibly accounting for up to 6% of the red cell volume. Very high Hcts, as in polycythemia, may also have excess plasma trapping. Manual Hct methods typically have a precision coefficient of variation (CV) of approximately 2%.

Automated analyzers do not depend on centrifugation techniques to determine Hct, but instead calculate Hct by direct measurements of red cell number and red cell volume (Hct = red cell number X mean red cell volume). Automated Hct values closely parallel manually obtained measurements, and the manual Hct is used as the reference method for hematology analyzers (wtih correction for the error induced by plasma trapping). Errors of automated Hct calculation are more common in patients with polycythemia or abnormal plasma osmotic pressure. Manual methods of Hct determination may be preferable in these cases. The precision of most automated Hcts is <1% (CV). 

Hemoglobin Concentration

Hemoglobin (Hb) is an intensely colored protein, allowing its measurement by spectrophotometric techniques. Hemoglobin is found in the blood in a variety of forms, including oxyhemoglobin, carboxyhemoglobin, methemoglobin, and other minor components. These may be converted to a single stable compound, cyanmethemoglobin, by mixing blood with Drabkin solution. Sulfhemoglobin is not converted but is rarely present in significant amounts. The main errors in measurement arise from dilution errors of increased sampel turbidity due to improperly lysed red cells, leukocytosis, or increased levels of lipid or protein in the plasma. With automated methods the precision for hemoglobin determinations is <1% (CV).

Red Cell Count

Manual methods for counting red cells have proven to be very inaccurate, and automated counters provide a much more accurate reflection of red cell numbers. Both erythrocytes and leukocytes are counted after whole blood dilution in an isotonic solution. As the number of red cells greatly exceeds the number of white cells, the error introduced by counting both cell types is negligible. However, when marked keukocytosis is present, red cell counts and volume determinations may be erroneous unless corrected for white cells. The observed precision for RBC counts using automated hematology analyzers is <1% (CV) compared with a minimum estimated value of 11% with manual methods.

Mean Corpuscular Volume

The MCV is usually measured directly with automated instruments but may also be calculated from the erythrocyte count and the Hct by means of the following formula:

MCV = Hct (L/L) X 1,000/red cell count (1012/L)

The MCV is measured in femtoliters (fl, or 10-15 L). Using automated methods, this value is derived by dividing the summation of the red cell volumes by the erythrocyte count. The CV in most automated system is approximately 1%, compared to ~10% for manual method. Agglutination of cells, as in cold agglutinin disease or paraproteinemia, may result in a falsely elevated MCV. Most automated analyzers gate out MCV values above 360 fl, thereyby excluding most cell clumps, although this may falsely lower Hct determinations. In addition, severe hyperglycemia (glucose >600 mg/dL) may cause osmotic swelling of the red cells, leading to a falsely elevated MCV.

Mean Corpuscular Hemoglobin

MCH is a measure of the average hemoglobin content per red cell. It may be calcuated manually or by automated methods using the following formula:

MCH = hemoglobin (g/L)/red cell count (1012/L)

MCH is expressed in picograms (pg, or 10-12 g). Thus, the MCH is a reflection of hemoglobin mass. MCH measurements may be falsely elevated by hyperlipidemia, as increased plasma turbidity will erroneously elevate hemoglobin measurement. Centrifugaton of the blood sample to eliminate the turbidity followed by manual hemoglobin determination allows correction of the MCH value. Leukocytosis may also spuriously elevate MCH values. The CV for automated analysis of MCH is <1% in most modern analyzers, compared with approximately 10% for manual methods.

Mean Corpuscular Hemoglobin Concentration

The average concentration of hemoglobin in a given red cell volume or MCHC may be calcualted by the following formula:

MCHC = hemoglobin (g/dL)/Hct (L/L)

The MCHC is expressed in grams of hemoglobin per deciliter of packed RBCs, representing the ratio of hemoglobin mass to the volume of red cells. With the exception of hereditar spherocytosis and some cases of homozygous sickle cell or hemoglobin C disease, MCHC values will not exceed 37 g/dL. This level is close to the solubility value for hemoglobin, and further increases in Hb may lead to crystallization. Factors that alert the accuracy of both Hct and hemoglobin can affect the precision of MCHC.

Red Cell Distribution Width

The red cell distribution width (RDW) is a red cell measurement that quantitates cellular volume heterogeneity reflecting the range of red cell sizes within a sample.

Reticulocyte Counts

Determination of the numbers of reticulocytes or immature, non-nucleated RBCs that still retain RNA provides useful information about the bone marrow's capacity to synthesize and release red cells in response to a physiologic challenge, such as anemia. In the past, reticulocyte counts were performed manually using supravital staining with methylene blue that will stain precipitated RNA as a dark blue meshwork or granules (at least two per cell), allowing retriculocytes to be identified and enumerated manually. Because there are relatively low numbers of reticulocytes, the CV for reticulocyte counting is relatively large (10% to 20%).

To increase accuracy of reticulocyte counting, automated detection methods to detect staining allow for many more cells to be analyzed, thereby increasing accuracy and precision of counts. Most of the newest automated hematology analyzers have automated reticulocyte counts to be included with routine complete blood count parameters. Reticulocytes are detected by a fluorescent dye that binds to RNA. Comparisons of stand-alone instruments and integrated hematology analyzers demonstrate superior accuracy when compared to manual counting methods, with CVs of 5% to 8%.


Update on Aug 2nd 2017

Ontogeny of Hemoglobin

The hemoglobin composition of the erythrocyte depends on when in gestation or postnatal development it is measured. This is a result of sequential activation and inactivation (i.e., switching) among genes within the alpha- and non-alpha-globin gene clusters. What controls these switches in globin gene transcription is not understood. The two early embryonic hemoglobins consist of ζ- and ε-globin chains (Hb Gower-1) and α- and ε-globin chains (Hb Gower-2). The ζ-globin gene is akin to the α-globin genes but is expressed only during early embryogenesis. The ε-embryonic globin chain is a β-like element. The combination of ζ- and γ-globin chains forms hemoglobin Portland. These early hemoglobins are made primarily in yolk-sac erythroblasts and are detectable only during the very earliest stages of embryogenesis except in certain pathologic states, in which they may persist until gestation is complete. The major hemoglobin of intrauterine life is HbF, which consists of two α- and γ-globin chains. Expression of the γ-globin gene begins early in embryogenesis, peaks during midgestation, and begins a rapid decline just before birth. By 6 months of age in normal infants, only a remnant of prior γ-globin gene expression remains. The level of HbF in the blood declines rapidly thereafter to less than 1% of the total. Expression of the α-globin gene starts early in the first trimester, peaks quickly, and is sustained for life. Expression of the β-globin gene also commences early in gestation and reaches its zenith within a few months after birth. The combination of α-globin with β-globin cahins forms hemoglobin A (HbA), the predominant hemoglobin of postnatal life. Adult cells also contain HbA2. The δ-globin gene, which directs synthesis of the non-α-globin chain of HbA2, is very inefficiently expressed. Only low levels of HbA2 are present; defects in the δ-globin gene are of no clinical consequence. In adult blood, HbF is not evenly distribbuted among erythrocytes and is present in only a very small number of RBCs, called F cells. HbA2 is present in all RBCs, albeit at levels less than 3.5% of the total hemoglobin in adult life.