Hematocrit

[Histology] The Blood Tissue

May 27, 2016 Hematology, Histology No comments , , , , , , , , , , , , , , ,

Major Componments of Blood

Blood is a specialized connective tissue in which cells are suspended in fluid extracellular material called plasma (ECM/extracellular matrix). When blood leaves the circulatory system, either in a test tube or in the ECM surrounding blood vessels, plasma proteins (coagulation factors) react with one another to produce a clot, which includes formed elements and a pale yellow liquid called serum. Serum contains growth factors and other proteins released from platelets during clot formation, which confer biological properties very different from those of plasma.

Collected blood in which clotting is prevented by the addition of anticoagulants can be separated by centrifugation into layers that reflect its heterogeneity. Erythrocytes make up the sedimented material and their volume, normally about 45% of the total blood volume in healthy adults, is called hematocrit. The straw-colored, translucent, slightly viscous supernatant comprising 55% at the top half of the centrifugation tube is the plasma. A thin gray-white layer called the buffy coat between the plasma and the hematocrit, about 1% of the volume, consists of leukocytes and platelets, both less dense than erythrocytes.

Function of the Blood Tissue

  • O2 is bound mainly to hemoglobin in erythrocytes and is much more abundant in arterial than venous blood, while CO2 is carried in solution as CO2 or HCO3, in addition to being hemoglobin-bound.
  • Nutrients are distributed from their sites of synthesis or absorption in the gut, while metabolic residues are collected from cells all over the body and removed from the blood by the excretory organs.
  • Hormone distribution in blood permits the exchange of chemical messages between distant organs regulating normal organ function.
  • Blood also participates in heat distribution, and the regulation of body temperature.
  • Blood maintain the acid-base and osmotic balance.
  • Blood can form clotting when bleeding happens, the hemostasis.
  • Leukocytes, complements, and antibodies have diversified functions and are one of the body's chief defenses against infection.

Plasma

Plasma is an aqueous solution, pH 7.35~7.45 (in normal conditions), containing substances of low or high molecular weight that make up 7% of its volume. The dissolved components are mostly plasma proteins, but they also include nutrients, respiratory gases, nitrogenous waste products, hormones, and inorganic ions (electrolytes). Through the capillary walls, the low-molecular-weight components of plasma (e.g., most drugs) are in equilibrium with the interstitial fluid of the tissues. Thus, the composition of plasma is usually an indicator of the mean composition of the extracellular fluids in tissues.

Major plasma proteins

  • Albumin, the most abundant plasma protein, is made in the liver and serves primarily to maintain the osmotic pressure of the blood.
  • alpha-Globulins and beta-globulins, made by liver and other cells, include transferrin and other transport factors; fibronectin; prothrombin and other coagulation factors; lipoproteins and other proteins entering blood from tissues.
  • gamma-Globulins, which are immunoglobulins (antibodies) secreted by plasma cells in many locations.
  • Fibrinogen, the largest plasma protein, also made in the liver, which, during clotting, polymerizes as insoluble, cross-link fibers of fibrin that block blood loss from small vessels.
  • Complement proteins, a system of factors important in inflammation and destruction of microorganisms.

RBCs

  • Human erythrocytes normally survive in the circulation for about 120 days. After 120 days, defects in the membrane's cytoskeletal lattice or ion transport systems begin to produce swelling or other shape abnormalities, as well as changes in the cells' surface oligosaccharide complexes. These RBCs are removed from the circulation mainly by macrophages ot the spleen, liver, and bone marrow.
  • Erythrocyte differentiation includes loss of the nucleus and organelles, shortly before the cells are released bone marrow into the circulation. Lacking mitochondria, erythrocytes rely on anaerobic glycolysis for their minimal energy needs.
  • The combination of hemolgobin with carbon monoxide (CO) is irreversible, howevver, reducing the cells' capacity to ransport O2 and CO2.

Leukocytes

Leukocytes leave the blood and migrate to the tissues where they become functional and perform various activities related to immunity. According to the type of cytoplasmic granules and their nuclear morphology, leukocytes are divided into two groups: granulocytes and agranulocytes. All granulocytes are terminally differentiated cells with a life span of only a few days. Their Golgi complexes and rough ER are poorly developed. They have few mitochondria and depend largely on glycolysis for their low energy needs. Granulocytes normally die by apoptosis in the connective tissue. The resulting cellular debris is removed by macrophages and, like all apoptotic cell death, does not itself elicit an inflammatory response.

  • Granulocytes: neutrophils, esoinophils, and basophils
  • Agranulocytes: lymphocytes and monocytes

Neutrophils

Mature neutrophils consitute 54% to 62% of circulating leukocytes; circulating immature forms raise this value by 3% to 5%. In females, the inactive X chromosome may appear as a drumstick-like appendage on one of the lobes of the nucleus although this characteristic is not obviious in every neutrophil. Neutrophils are inactive and spherical while circulating but become actively amoeboid during diapedesis and upon adhering to solid substrates such as collagen in the ECM.

Neutrophils are active phagocytes of bacteria and other small particles and are usually the first leukocytes to arrive at sites of infection, where they actively pursue bacterial cells using chemotaxis. The cytoplasmic granules of neutrophils provide the cells' functional activities and are of two main types (primary granules and secondary granules). Azurophilic primary granules resemble lysosomes as large, dense vesicles and have a major role in both killing and degrading engulfed microorganisms. They contain proteases and antibacterial proteins, including: 1.myeloperoxidase/MPO, which generates hypochlorite and other agents toxic to bacteria; 2.lysozyme, which degrades components of bacterial cell walls; and 3.defensins, small cysteine-rich proteins that bind and disrupt the cell membranes of many types of bacteria and other microorganisms. Specific secondary granules are smaller, less dense, and have diverse functions, including secretion of various ECM-degrading enzymes such as collagenases, delivery of additional bactericidal proteins to the phagolysosomes, and insertion of new cell components.

Activated neutrophils at infected or injured sites also have important roles in the inflammatory response, including the release of chemokines that attract other leukocytes; cytokines that direct activites of these and local cells of the tissue; and the release of lipid mediators of inflammation.

Neutrophils are short-lived cells with a half-life of 6 to 8 hours in blood and a life span of 1 to 4 days in connective tissues before dying by apooptosis.

Eosinophils

Eosinophils are far less numerous than neutrophils, constituting only 1% to 3% of leukocytes. The main identifying characteristic is the abundance of large, acidophilic specific granules typically staining pink or red. Ultrastructurally the eosinophilic specific granules are seen to be oval in shape, with flattened crystalloid cores containing major basic protein/MBP, an arginine-rich factor that act to kill parasitic worms or helminths. Eosinophils also modulate inflammatory responses and allergies.

Basophils

The specific granules in basophils contain heparin and other sulfated GAGs, much histamine and various other mediators of inflammation. By migrating into connective tissues, basophils appear to supplement the function of mast cells. Like mast cells, basophils secretion these granules in response to certain antigens and allergens.

Lymphocytes

By far the most numerous type of agranulocyte in normal blood smears of CBCs, lymphocytes constitute a family of leukocytes with spherical nuclei. Although they are morphologically similar, lymphocytes can be subdivided into functional groups by distinctive surface molecules (called "cluster of differentiation" or CD markers) that can be distinguished using antibodies wtih immunocytochemistry or flow cytometry. Major classes include B lymphocytes, helper and cytotoxic T lymphocytes (CD4+ and CD8+, respectively), and natural killer (NK) cells.

Lymphocytes vary in life span according to their specific functions; some live only a few days and others survive in the circulating blood or other tissues for many years.

Monocytes

Monocytes are agranulocytes that are precursor cells of macrophages, osteoclasts, microglia, and other cells of the mononuclear phagocyte system in connective tissue. All monocyte-derived cells are antigen-presenting cells and have important roles in immune defense of tissues.

Platelets

Blood platelets are very small non-nucleated, membrane-bound cell fragments only 2 to 4 um in diameter. Platelets originate by separation from the ends of cytoplasmic processes extending from giant polyploid bone marrow cells called megakaryocytes. Platelets promote blood clotting and help repair minor tears or leaks in the wall of small blood vessels, preventing loss of blood from the microvasculature. Circulating platelets have a life span of about 10 days.

A sparse glycocalyx surrounding the platelet plasmalemma is involved in adhesion and activation during blood coagulation. The role of platelets in controlling blood loss and in wound healing can be summarized as follows:

  • Primary aggregation: Disruptions in the microvascular endothelium, which are very common, allow the platelet glycocalyx to adhere to collagen. Thus, a platelet plug is formed as a first step to stop bleeding.
  • Secondary aggregation: Platelets in the plug release a specific adhesive glycoprotein and ADP, which induce further platelet aggregation and increase the size of the platelet plug.
  • Blood coagulation: During platelet aggregation, fibrinogen from plasma, von Willebrand factor and other protein released from the damaged endothelium, and platelet factor 4 from platelet granules promote the sequential interaction of plasma proteins, giving rise to a fibrin polymer that forms a three-dimensional network of fibers trapping red blood cells and more platelets to form a blood clot, or thrombus. Platelet factor 4 is a chemokine for monocytes, neutrophils, and fibroblasts and proliferation of the fibroblasts is stimulated by PDGF.
  • Clot retraction: The clot that initially bulges into the blood vessels lumen contracts slightly because of the interaction of platelet actin and myosin.
  • Clot removal: Protected by the clot, the endothelium and surrounding tunic are restored by new tissue, and the clot is then removed, mainly dissolved by the proteolytic enzyme plasmin, formed continuously through the local action of plasminogen activators from the endothelium on plasminogen from plasma.

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.