Erythropoietin (EPO)

The role of red blood cells is to carry oxygen. Just like anything in the body, this is tightly regulated by a mechanism that monitors whether or not there is adequate oxygen getting to tissues and other cells. Hypoxia is detected by the peritubular fibroblasts of the kidneys which causes erythropoietin (EPO) to be released. The EPO gene has a hypoxia-sensing region in its 3’ regulatory component which causes hypoxia inducible factor-1 (HIF-1) transcription factor to be assembled and it interacts with the 3’ enhancer of the gene causing increased EPO mRNA and production of more EPO.  EPO is a true hormone, being produced in the kidneys, and acting upon another distant location being the bone marrow. When EPO binds to its ligand (receptor) on red blood cell progenitors it initiates a cascade which is mediated through the JAK2 signal transducers which ultimately effects the gene expression. EPO has three main physiological effects on the body; it allows early release of reticulocytes from the bone marrow, prevents apoptosis, and reduces the time needed for cells to mature in the bone marrow before release into the periphery. 

There are two mechanism for which EPO stimulates early release of red cell precurors into the bone marrow. It induces changes in the adventitial cell layer of the marrow sinuses that increases the width of the spaces that the red cells squeeze out of. It also down regulates red blood cell surface receptors for adhesive molecules that are located on the bone marrow stroma. As a result the red cells are able to pass through without the receptor so that they won’t bind to the stroma and delay release.

Apoptosis is programmed cell death. EPO inhibits apoptosis by removing the induction signal. Under normal physiology the bone marrow produces more CFU-Es than needed that are stored in the bone marrow which have a “head start” in the maturation process. About a 10 day head start in maturation. The CFU-Es (Colony-forming unit-erythroid) are red blood cell progenitor cells that develop from BFU-Es (Burst-forming unit-erythroid). Both BFU-E and CFU-E are red blood cell progenitor cells that develop into the pronormoblast, which is the first morphologically identifiable red blood cell precursor. If healthy, those cells live out there life span and undergo apoptosis. If there is a deficiency of red blood cell mass, those cells undergo maturation to be released, while simultaneously the apoptosis induction signal is inhibited. The normal death signal consists of a death receptor being FAS, on the membrane of the earliest red blood cell precursors (CFU-Es/BFU-Es), and FASL ligand on the maturing red blood cells precursors. When EPO levels are low, because there is adequate oxygen delivery the older FASL bearing cells cross-link with earlier FAS precursors which stimulates apoptosis. EPO is able to subdue apoptosis by stimulating the more mature precursors to be released from the marrow, especially in times of hypoxia. At which point there will no FASL bearing cells to cross-link the early FAS bearing precursors. Its a two fold effect, the more mature cells are released to help increase red cell mass in times of need, and the early precursor are allowed to mature and be released without undergoing apoptosis. When EPO binds to its ligand on the red blood cell activates the JAK2-STAT pathway, which ends in and up-regulation of transcription for BCL-2, which is an anti-apoptotic protein. This anti-apoptotic protein rests on the cell membrane and prevents the release of cytochrome c, which initiates apoptosis. 

JAKSTAT

EPO has an effect on the bone marrow transit time of a red blood cell precursor in two different ways; increased rate of cellular processes, and decreased cell cycle times. What this means is that EPO stimulates synthesis of red cell RNA, such as the production of hemoglobin. It also stimulates the production of egress-promoting surface molecules within the bone marrow which allow the red blood cells to flow through the marrow easier. EPO stimulates cells to enter cell cycle arrest earlier than normal, and as a result, spend less time maturing and are able to be released. These cells may appear larger in size and have a bluish tinge to their cytoplasm because of this.

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Laboratory Equation Guide

SI Units: The International System of Units is a system devised around the convenience of the number ten. It is the worlds most widely used system of measurement in science.

Definitionsandabbreviations

Temperature

SI Standard units are Kelvin (K)

Celsius = K-273

Kelvin = Celsius+273

Temperature Conversions

Celsius = 5/9 (F – 32)

Fahrenheit = (9/5 x C) + 32

Solution

A solution is a homogenous mixture of two or more substances. A solute is a substance that is small part of a solution. A solvent is a substance that constitutes a large portion of a solution. Solubility refers to the solutes ability to dissolve in the solvent.

Dilutions 

A dilution is when the solute or substance of interest is combined with an appropriate volume of solvent to achieve a desired concentration. The dilution factor is the resulting total number of unit volumes in which the solute was dissolved.

For example: Dissolving one part solute into 3 parts solvent. Total dilution is 1:4. The dilution factor is 4 because there are four total parts of unit volumes.

(C1)(V1)=(C2)(V2) is used when making fixed volumes of specific concentrations.

Percent solutions refer to parts per hundred. They can either be percent by volume (% (v/v)) or percent by mass (% (m/m)).

Percent by Volume (% (v/v)) = (Volume of solute/Volume of solution) x 100

Percent by Mass (% (m/m)) = (Mass of solute/Mass of solution) x 100

Molarity is a unit of concentration equal to the number of moles of solute in one liter of solution.

Molarity (M) = Moles of solute/Liters of solution

Density is the ratio between the mass and volume of a material.

Density = Mass/Volume

Clinical Validity

Specificity: The frequency of a negative test when no disease is present.

Specificity = (True negatives/True negatives+False positives) x 100

Sensitivity: The frequency of a positive test when disease is present.

Sensitivity = (True positives/True positives+False negatives) x 100

Chemistry

Calculated plasma osmolality = 2[Na+] + Glucose/18 + BUN/2.8

Osmolar Gap: Difference between the measured and calculated osmolality.

Friedman Formula:

LDL = Total cholesterol – HDL – VLDL

Triglycerides = (Total cholesterol – HDL – LDL) x 5

Precaution: The Friedman formula is only reliable for triglyceride levels less than 400 mg/dL.

Bilirubin

Total Bilirubin = Conjugated bilirubin + Unconjugated bilirubin

Conjugated Bilirubin = Total bilirubin – Unconjugated bilirubin

Unconjugated bilirubin = Total bilirubin – Conjugated bilirubin

Creatinine Clearance

Cockcroft-Gault Equation

Creatinine Clearance = Male (1.0), Female (0.85) x (140-age) x (Serum Creatinine) x (Weight/72).

Hematology

Hematocrit: The percentage of blood that is represented by packed red cells.

Hematocrit (%) = Hemoglobin x 3

MCV: Mean cell volume refers to the average size of the red cell population within the sample.

MCV = (Hematocrit (%) x 10)/RBC (x10^12/L)

MCH: Mean cell hemoglobin refers to the average weight of hemoglobin within the red cell population.

MCH = (Hgb x 10)/RBC (x10^12/L)

MCHC: Mean cell hemoglobin concentration refers to the average concentration of hemoglobin within the red cells constituting the sample.

MCHC: (Hgb x 100)/Hematocrit (%)

Corrected WBC: Nucleated RBCs are counted as white blood cells regardless of which method is utilized. For this reason when a differential is performed and there is presence of NRBCs a corrected WBC must be calculated. The number of NRBCs per 100 leukocytes is recorded during the differential leukocyte count when performing a blood smear examination. This number is then used;

Corrected WBC = (WBC x 100)/(NRBC + 100)

 

 

 

 

Thalassemias

PIT11

The major hemoglobin that is present in adults is hemoglobin A (HbA). This is a heterotetramer that consists of one pair of alpha-globin chains and one pair of beta-globin chains. Alpha-globin chains are encoded by two copies of the alpha gene present on chromosome 16. Beta-globin chains are driven by one gene on chromosome 11. Normally there is tight regulation of the production of alpha and beta-globin chains and the ratio of production, but sometimes that regulation can be interrupted. This offsets the balance of globin chains being produced. These types of hematological disorders are coined thalassemias. They are a quantitative defect characterized by reduced or absent production of one and rarely two of the globin chains.

Alpha thalassemia is largely due to the inadequate production of alpha globin chains, which leads to an excessive production of either gamma-globin chains or beta-globin chains. In the fetus alpha thalassemia leads to excess gamma chains and in adults it largely leads to excess beta chains. In neonates the absence of alpha-globin chains is incompatible with life, leading to hydrops fetalis or hemoglobin Barts and absolute death after delivery. Hb Barts cannot deliver the oxygen to the tissues because its affinity to oxygen is too high. The hydronic state is reflected in the fetus by heart failure and massive total body edema. Excess beta-globin chains are capable of forming homotetramers and precipitate that leads to a variety of clinical manifestations.

Beta thalassemia is an inherited hemoglobinopathy in which production of beta-globin chains is impaired. There are different classifications corresponding to the degree of reduction in the beta chains. Beta thalassemia major is due to mutations that completely stop all production of beta-globin chains. These are individuals who are homozygous for the disease. They lose the ability to make HbA and because of this will experience severe manifestations and are transfusion-dependent for the rest of life. Symptoms typically begin during late infancy (6-12 months), but some newborns are asymptomatic because the major hemoglobin in newborns is HbF (4A:4G) which is constructed by gamma-globin chains and not beta-globin chains. Beta-thalassemia major presents with pallor, jaundice, and bilirubin in the urine which indicates hemolysis. Hepatosplenomegaly is present as well as heart failure. Failure to thrive and recurrent infections are also other signs. There is so much hemolysis because of the faulty hemoglobin present in the red cells that the bone marrow can’t keep up with production so extra medullary hematopoiesis occurs that results in skeletal abnormalities in the face and long bones. Iron overload is often a symptom of late untreated disease which can affect almost every organ in the body. Mortality is upwards of 85% by age five if untreated. If treated the survival rate is only 60 years of age if lucky.

Beta thalassemia major is also called transfusion-dependent beta thalassemia. There is also a subtype called non-transfusion-dependent beta thalassemia otherwise known as beta thalassemia intermedia. These individuals present with a less severe phenotype of the disease. There is significant variability with the clinical findings in individuals with beta thalassemia intermedia; from osteoporosis to thrombosis to diabetes mellitus. Some individuals will develop hepatosplenomegaly and extramedullary hematopoiesis and some won’t. Also some individuals will have to become transfusion-dependent, but that is typically in the late decades of life.

Anemia is a severe clinical manifestation of both alpha and beta thalassemia. The pathophysiology of beta thalassemia causes excess alpha-globin chains to precipitate in the developing erythrocytes in the bone marrow. This causes inclusion bodies. The inclusion bodies create oxidative stress and damages the cellular membranes. Apoptosis gets activated downstream and the red cell precursors are subsequently phagocytized and destroyed in the bone marrow by activated macrophages. This is also called ineffective erythropoiesis. The bone marrow in an effort to compensate releases these red cell precursors into the peripheral blood riddled with these inclusion bodies. These cells are subsequently sequestered by extravascular hemolysis by the RES which  further contributes to the anemia. The red cells that survive are microcytic and hypochromic and have a significantly shortened life span. Severe tissue hypoxia is seen due to the increased HbF as a compensatory mechanism. HbF has an increased affinity for oxygen and causes a shift to the left on the oxygen dissociation curve.

Typical laboratory findings for an individual with beta thalassemia is a slightly decreased red cell count and a marked decrease in hemoglobin of usually about 2-3 g/dL (12.5-16.5 g/dL). There will be marked anisocytosis (microcytosis) and poikilocytosis, target cells, basophilic stippling, slight increase in reticulocytes and nucleated red cells.

The pathophysiology for anemia associated with alpha thalassemia is associated with precipitation of HbH. Remember HbH is formed when there is decreased production of the alpha-globin chains so there is an excess of beta-globin chains. The precipitation of HbH creates inclusion bodies, typically called Heinz Bodies. These inclusions are recognized by the RES and remove the red cells via extravascular hemolysis.

Laboratory findings for an individual with alpha thalassemia is very similar to that of an individual with beta thalassemia. Decreased hemoglobin, marked anisocytosis (microcytosis) and poikilocytosis, target cells, basophilic stippling, and reticulocytes and NRBCs. The HbH inclusions can be see seen using a cresyl blue stain.

Thalassemias are a quantitative hemoglobinopathy meaning that there is a deficiency or an excess of production of globin chains leading to clinical manifestations. They are inherited and some subtypes can significantly elevate mortality. It is important to diagnose early and to treat early.

 

 

 

Non-Malignant Leukocyte Disorders

Non-Malignant simply means that it is localized to the leukocytes. Leukocytes are another name for the white blood cells, more specifically in the case of these disorders, the granulocytes. These disorders are fairly uncommon and are inherited. The following are ones that are found to distinct morphological features and affect the granulocyte functionality.

Alder Reilly Anomaly

Alder Reilly Anomaly is a recessive trait defect that causes incomplete degranulation of mucopolysaccharides. Large, darkly staining metachromatic cytoplasmic granules which can be seen and are partially digested mucopolysaccharides. These granules are characteristically referred to as Alder Reilly bodies. These can sometimes resemble toxic granulation, but it is important to note that in toxic granulation neutropenia, dohle bodies, and a left shift is seen. In Alder Reilly Anomaly none of those are present. Its also important to mention that the functionality of the granulocytes is not impaired.

Alder Reilly

Pelger Huet Anomaly

Pelger Huet Anomaly is an autosomal dominant syndrome characterized by decreased nuclear segmentation. This is caused by a mutation in the Lamin B receptor gene. Lamin B is an inner nuclear membrane protein that plays a role in normal leukocyte nuclear shape change during maturation. Morphological changes include hyposegmented neutrophils or neutrophil lobes connected by a thin nuclear filament. Pseudo or acquired PHA can be observed in the granulocytes in individuals with MDS, AML, or chronic myeloproliferative neoplasms.

Pelger Huet

Chediak Higashi Syndrome

Chediak Higashi Syndrome is characterized by an abnormal fusion of granules. These present as large and are dysfunctional. This is caused by a mutation in the LYST, or CHS1 gene that encodes for proteins involved in vesicle fusion or fission. The mutated protein causes loss of lysosomal movement and loss of phagocytosis. Thus leaving the individual susceptible to an increased number of infections without the innate immune system to fight them off. One of the characteristic findings is neutropenia.

CHA

May-Hegglin Anomaly

May-Hegglin Anomaly is a rare autosomal dominant platelet disorder that is characterized by variable thrombocytopenia, giant platelets, and dohle bodie like inclusions in the granulocytes. MHA is caused by a mutation in the MYH9 gene that causes a dysfunctional and disarray production of myosin heavy chains type IIa which affects the megakaryocytic maturation process as well as platelet fragmentation. Though most cases are clinically asymptomatic, the individual may present with mild bleeding tendencies.

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Chronic Granulomatous Disease

In CGD, mutations in proteins that make up the NADPH oxidase complex. The mutations lead to failure of the phagocytes to generate the oxygen-dependent respiratory burst following phagocytosis. Normal phagocytosis of a microorganism leads to phosphorylation of cytosolic P47 and P67. Antibacterial neutrophil elastase and cathepsin G from the primary granules and cytochrome complex gp91 and gp22 from the secondary granules migrate to the phagolysosome. NADPH oxidase is formed when P47 and P67 combine with P40, RAC2, and the cytochrome complex. Majority of cases of CGD is due to mutations in P47 or gp91.

Leukocyte Adhesion Disorders

Normal recruitment of leukocytes to a site of inflammation involves capture of leukocytes from peripheral blood, followed by a process known as rolling along a vessel wall. Rolling involves binding of integrins to endothelial cell receptors which is high-affinity which ultimately leads to diapedesis of leukocytes into tissues from peripheral blood. With Leukocyte Adhesion disorders there are mutations that result in the inability of neutrophils and monocytes to adhere to endothelial cells, and the consequence is potentially fatal bacterial infections.

Leukocyte Adhesion Disorder I is caused by a mutation in the genes responsible for B2 integrin subunits. This leads to a decreased amount of the truncated form of the B2 integrin which is essential for endothelial cell adhesion. Patients typically present with neutrophilia, lymphadenopathy, splenomegaly, and characteristic skin lesions.

Leukocyte Adhesion Disorder II is caused by a mutation in the SLC35C1 gene. This gene encodes for a fucose transporter that moves fucose from the endoplasmic reticulum to the Golgi region. Fucose is needed for the synthesis of selectin ligands. The defective fucose transporter leads to the inability to produce functional selectins and causes defective leukocyte recruitment and reoccurring infections. LADII is much more rare than LADI. Clinical presentation is growth retardation, coarse facial features, and other physical deformities.

Leukocyte Adhesion Disorder III is even more rare than LADII and is caused by a mutation in the Kindlin-3 gene. The mutations impair leukocyte rolling and activation of B integrin. With LADIII there is also decreased platelet integrin GPIIbIIIa resulting in bleeding similar to that of Glanzmann Thrombasthenia.

-Caleb

Blood Draw Tube Colors and Order

The tube order may not seem like a big deal and may seem unnecessary to some, but it is very important to pay attention too. It also matters as to what type of needle is being used for the draw. If a butterfly needle is being used it is important to have a spit tube because with a butterfly there is air within the hose that connects the needle to the vacutainer. Its important to get this air out before filling any tubes used for patient testing. If a standard needle is being used, you typically don’t need the spit tube, but its good practice. The order still remains the same for each.

Light Blue: The typical tube for routine coagulation studies. The additive is sodium citrate (3.2% or 3.8%). Citrate is a anticoagulant which binds to calcium within the blood so the blood can’t clot. Calcium plays an important role in primary and secondary homeostasis. See my post on DIC for that information, in short it is used in the coagulation cascade. An important aspect of coagulation studies is that the light blue tube must, must be filled completely. There is a ratio of sodium citrate to whole blood and that must remain constant. The tube must be rejected if it is not filled completely.

Green or Mint Tubes: These tubes are used for chemistry studies. Often referred to as PST or plasma separator tubes. The additive in these tubes are sodium heparin, lithium heparin or ammonia heparin. The heparin, being an anticoagulant activates antithrombin, which blocks the coagulation cascade and produces a whole blood with plasma sample instead of a clotted blood and serum sample. When these tubes are centrifuged, the gel barrier moves upwards creating a barrier that separates the plasma from the red cells allowing the plasma to be aspirated directly for testing.

Gray Tube: The gray tube tops are typically used for glucose testing, ethanol levels or lactate level testing. The additive is potassium oxalate and sodium fluoride. Potassium oxalate is an anticoagulant which prevents clotting and the sodium fluoride is an anti-glycolytic which prevent the cells from using the glucose in the sample.

Lavender/Pink Tube: The lavender tube is typically used for hematological testing or for Hemoglobin A1C testing. The pink tube is used primarily for blood bank testing such as type and screen and cross-matching. The additives in the lavender and pink tubes are EDTA K2 or EDTA K3. The EDTA binds to calcium which blocks the coagulation cascade in the same way that citrate in the light blue tube does. Red cells, leukocytes, and platelets are in EDTA anticoagulated blood for 24 hours. Blood smears should be done within 3 hours of receiving the sample.

SST/Mustard Tube: Serum separator and clot activator that will separate the blood from the serum upon centrifugation. This tube is usually used to test for aldosterone, B12, ferritin and folate levels.

There are not all the different tubes that are used, but these are the most common tubes that I listed and the ones that a laboratory professional will most likely come across. Its important to understand the additive in each one to make sure that they are appropriate for the testing that needs to be done on the patient sample itself within the tube.

phb_Order of Blood Draw with labels72dpi

-Caleb