Glycolysis

Glycolysis is the first phase of a series of reactions for the catabolism of carbohydrates. Catabolism is the breakdown of larger molecules into its respective smaller constituents. Glycolysis is the first part of cellular respiration that generates pyruvate to be used in either anaerobic respiration in the absence of oxygen or in the TCA cycle in aerobic respiration which yields useable energy for cells. This will be a general outline of the steps in glycolysis.

The whole process can be broken down into an energy investment phase where ATP is being used and an energy payoff phase where ATP is being generated. Fructose-1,6-biphosphate is where the energy investment phase ends. That is where the last ATP has to be used for energy to drive glycolysis.

A simple equation can be remembered as a summary of glycolysis.

Glucose + 2 ADP + 2 phosphate ions + 2 NAD+ —-> 2 Pyruvate + 2 ATP + 2 NADH + 2 H20 + 2 H+.

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In the first step of glycolysis an addition of a high energy phosphate from ATP yields glucose-6-phosphate and ADP. This step is initialized by the enzyme hexokinase. G6P is more reactive than glucose.

In the next step, glucose-6-phosphate is converted to its isomer, fructose-6-phosphate by phosphoglucose isomerase.

In the third step of glycolysis, fructose-6-phosphate is converted to fructose-1,6-biphosphate by phosphofructokinase (PFK) and the addition of ATP. This is the committed step, meaning that fructose-1,6-biphosphate MUST be converted to pyruvate. This is also the end of the energy investment phase of glycolysis.

Fructose-1,6-biphosphate is converted to glyceraldehyde-3-phosphate (G3P) and dihydroxyacetone phosphate catalyzed by aldolase. Glyceraldehyde-3 phosphate maintains a reversible reaction with dihydroxyacetone phosphate through triode phosphate isomerase. The resulting reaction generates two molecules of glyceraldehyde-3-phosphate. A key point going forward is that two molecules of each substrate are produced.

Each G3P molecule gains an inorganic phosphate and with the addition of NAD+ to form the energized carrier molecules NADH. The resulting reaction catalyzed by glyeraldehyde-3-phosphate dehydrogenase generates two molecules of 1,3-bisphophoglycerate which yield two high energy phosphates.

Through the addition of two low energy ADP molecules and the enzyme phosphoglycerate kinase, the two molecules of 1,3-bisphophoglycerate are converted to 3-phosphoglycerate and yields two molecules of ATP. This reaction is called the break even reaction because at this point the energy input is equal to the energy output. Two molecules of ATP were expended and at this step there was a generation of two ATP molecules.

In the next step the two molecules of 3-phosphoglyercate are converted to 2-phosphoglycerate through the enzymatic properties of phosphoglycerate mutase.

The molecules of 2-phosphoglycerate are converted to phosphoenolpyruvate catalyzed by enolase. This step yields H20 molecules.

In the final step of glycolysis, the molecules of phosphoenolpyruvate are converted to pyruvate catalyzed by pyruvate kinase. ATP is generated from the addition of ADP and the two high energy phosphates from the molecules of phosphoenolpyruvate.

Upon the completion of glycolysis, the pyruvate molecules can be oxidized to carbon dioxide in cellular respiration to generate 28 molecules of ATP.

The NADH that is produced is turned back into NAD+ to drive further glycolysis. There are two ways to accomplish this. In the presence of oxygen NADH passes it electrons into the electron transport chain, which regenerates NAD+ for use in glycolysis. In the absence of oxygen, cells regenerate NAD+ by undergoing fermentation.

 

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Blood Components 101

This will serve as a guide for the specific indications, storage requirements and stability of the different blood components.

Storage

Whole Blood, Packed Red Blood Cells: 1-6 degrees Celsius.

Plasma, Cyroprecipitated AHF: -18 degrees Celsius.

Platelets: 20-24 degrees Celsius with continuous gentle agitation.

Granulocytes: 20-24 degrees Celsius without agitation.

Stability

Whole Blood: When refrigerated a unit of whole blood has a shelf life between 21-35 days depending on the additive that is used. Must be transfused within 4 hours when at room temperature.

Packed Red Cells: Packed red cells are stable for up to 42 days refrigerated, but they can also be frozen with glycerol as a cyroprotectant for up to 10 years. They must be deglycerolized by washing and thawed prior to transfusion and must be transfused within 24 hours once thawed.

Platelets: Platelets have a shelf live of only 5 days. Some hospitals and clinics are extending the shelf life out to 7 days with continuous bacterial testing to ensure there is no contamination.

Plasma: Plasma products must be processed and frozen within 8 hours of collection and are stable for 12 months. Once thawed they must be transfused within 24 hours.

Thawed Plasma: Has an expiration of 5 days.

Cryo: Cyro AHF once pooled and frozen has a stability of up to 12 months.

Granulocytes: Granulocytes must be transfused within 24 hours after donation.

Constituents

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Whole Blood: Used to replace the loss of both RBC mass and plasma volume. The product is 550-600 mL of whole blood, with a hematocrit of about 40%.

Packed Red cells: Usually the red cell product of choice. 330 mL of red cells, hematocrit of about 55-65% with an additive solution.

 

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Platelets: Platelets derived from whole blood must contain at least 5.5×10^10 platelets in 40-70 mL of plasma in at least 90% of the units tested. Platelets donated through apheresis must contain at least 3×10^11/L platelets in 100-500 mL of plasma. One apheresis platelet collection is equivalent to six pooled random donor platelet concentrates.

 

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Plasma: Can be derived from whole blood or apheresis collection. Plasma contains albumin, coagulation factors, fibrinolytic proteins, and immunoglobulins. Fresh frozen plasma (FFP) derived from whole blood is usually 220-300 mL and units derived from apheresis usually contain 400-600 mL. The plasma must be frozen within 8 hours of collection.

 

Final_Blood_products

Cryoprecipitated Antihemophilic Factor (AHF): AHF is prepared from FFP. It is slowly thawed, then refrozen within one hour of thawing. AHF typically contains 5-20 mL of plasma with 80-120 U/concentrate of Factor VIII, 150-250 mg/concentrate of fibrinogen, 40-70% of vWF, and 20-30% of Factor XIII that would normally be present in FFP. Making it the treatment of choice for Von Willebrands Disease and Hemophiliacs.

Indications

Red Cells

Red cell transfusions are used to treat hemorrhage and to improve oxygen delivery to tissues. The decision to transfuse red cells should be based on the patients clinical condition. Indications for red cell transfusion include acute sickle cell crisis, acute blood loss of greater than 30% of blood volume, or patients with symptomatic anemia that can’t function without red cell repleting. The threshold for transfusion of red cells should be a hemoglobin of 7 g/dL in adults and children. Maintenance can be at a level of >7-9 g/dL.  One unit of red cells should increase the hemoglobin by 1 g/dL and hematocrit by 3%.

Washed Red Cells

Washed red cells are washed with saline to remove any residual plasma proteins. These are used for patients with a history of allergic transfusion reactions. These patients have an IgA deficiency and have developed anti-IgA.

Leukocyte Reduced

Leukocyte reduced red cells decrease the incidence of febrile transfusion reactions. They are indicated for those at high risk of transfusion-associated GVHD or transfusion-related immune suppression. For a unit to be considered leukocyte reduced, there must be less than 5×10^6 leukocytes.

Irradiated Red Cells

Used for patients with a history of febrile transfusion reactions or patients that are immunocompromised immediately after an allogeneic bone marrow or stem cell transplant. Patients at risk for HLA-GVHD will receive irradiated red cells.

Plasma

Plasma transfusion are recommended for patients with active bleeding and an international normalized ratio (INR) greater than 1.6. Its indicated for patients on anticoagulant therapy that are undergoing an invasive procedure. Plasma should not be administered for a high INR without active bleeding. Plasma is indicated for patients with inherited clotting factor deficiencies for which there is no safe recombinant factor available. Those factors are II, V, X, and XI. Plasma is used as an emergent reversal of warfarin (coumadin) toxicity to prevent intracranial hemorrhage. It is also used in acute disseminated intravascular coagulation (DIC) or other thrombotic microangiopathies such as thrombotic thrombocytopenic purpura (TTP) or hemolytic uremic syndrome (HUS). Plasma is often times transfused with red cells during massive transfusions; with the definition of massive transfusion being greater than 5,000 mL in an adult of average weight (70 kg).

Platelets

Platelet transfusions are indicated to prevent hemorrhage in patients with thrombocytopenia or those with functional platelet defects. Contradictions for platelet therapy are patients with TTP and heparin-induced thrombocytopenia (HIT) as transfusion in these clinical situations can result in exacerbation of thrombosis. Platelet transfusions can be used prophylactically in invasive surgeries with no active bleeding and commonly used in active bleeding situations along with transfusion of FFP and red cells. One unit of apheresis platelets should increase the platelet count in adults by 30-60×10^9/L.

Transfusion of neonates is complicated and should be based on upon clinical reasons with consideration to the platelet count. If the count is <20×10^3/mL, you should always transfuse if possible. When you reach 20-30×10^3/mL you should consider transfusion, but weigh all possibilities. In a case of active bleeding, transfusion is absolutely appropriate, but all factors should be considered. Transfusion is also indicated in there is signs of a coagulation disorder, intraventricular or intraparenchymal cerebral hemorrhage, an invasive procedure, or if there is alloimmune neonatal thrombocytopenia.

Cryoprecipitate AHF

Cryo contains high concentrations of factor VIII and fibrinogen and is used especially in cases of hypofibrinogenemia. Hypofibrinogenemia is typically seen in the setting of massive hemorrhage or in a consumptive coagulopathy such as DIC. Indications for cyroprecipitate AHF are factor VIII and factor XIII deficiency, congenital fibrinogen deficiency, and von Willebrand disease.

 

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Granulocyte Pheresis

Indicated for patients with fever, neutropenia, septicemia or an antibiotic resistant bacterial infection.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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)

 

 

 

 

Serum Protein Electrophoresis (SPE)

Protein electrophoresis measures the specific proteins in the body by using electrical charge to separate them. Serum proteins are either albumin or they are globulins. Globulins are then further delegated into either gamma globulins or alpha-1, alpha-2, or beta globulins.

The normal ranges for each protein fraction in the serum is as follows;

Albumin: 3.6-5.2 g/dL

Alpha-1: 0.1-0.4 g/dL

Alpha-2: 0.4-1.0 g/dL

Beta: 0.5-1.2 g/dL

Gamma: 0.6-1.6 g/dL

Normal SPE

SPE is useful in the diagnosis of Multiple Myeloma or Waldenstrom macroglobulinemia which presents as a characteristic elevation in the gamma globulin peaks. It can also be used to help diagnose liver diseases, renal diseases, anemia, and even malnutrition.

Based upon which serum protein fraction is either decreased or increased gives evidence to a specific diseases or a group of diseases.

Albumin is produced by the liver and is the most abundant and arguably one of the most important proteins in the body. One of its main functions is to maintain the colloid pressure between the tissues and the bloodstream. Increases in albumin are seen in severe dehydration. They are decreased in malnutrition, liver disease, nephrotic syndrome or severe burns.

The major alpha-1 globulin is alpha-1 antitrypsin, which is produced in the lungs and in the liver. Increases in alpha-1 are seen in inflammatory states and pregnancy. Alpha-1 is decreased in alpha-1 antitrypsin deficiency. Alpha-1 antitrypsin deficiency is used as marker for an increased risk of emphysema.

Alpha-2 globulins include serum haptoglobin, alpha-2-macroglobulin, and ceruloplasmin. Haptoglobin binds to any free hemoglobin in the blood as a result of intravascular hemolysis to prevent its excretion by the kidneys. Ceruloplasmin is the major protein in the body that carries copper which also plays a role in iron metabolism. Increases in alpha-2 are also seen in inflammatory states and nephrotic syndromes. In cases of nephrotic syndromes you will see a decrease in albumin and a compensatory increase in alpha-2. Alpha-2 may also be elevated in hyperthyroidism, steroid use, and oral contraceptives. It is usually decreased in hemolysis and liver disease.

Beta-globulins include transferrin, low-density lipoproteins (LDL) and complement proteins. Transferrin is the molecule that is used to transport dietary iron to the liver, spleen and bone marrow for storage. LDL is the major carrier of cholesterol in the blood. Complement is a branch of the immune system that plays a specific role in the inflammatory response. Beta protein fractions are increased in hyperlipidemia and iron deficiency anemia. It is decreased in malnutrition.

Gamma-globulins encompass the different classes of immunoglobulins. Gamma globulins are increased in either monoclonal or polyclonal gammopathies. It is decreased in agammaglobulinemia and hypogammaglobulinemia. Immunoelectrophoresis is a reflex test that detects the levels of the different immunoglobulins within the gamma-globulin which should be used when there is an abnormal amount of protein detected.

serum electrophoresis with molecules included

A monoclonal gammopathy is a narrow band increase in the gamma protein fraction that is composed of a single class of immunoglobulins secreted by a malignant clone of plasma cells. It is also known as the M-protein. M-protein is typically representative of a diagnosis of multiple myeloma, but can be detected in other lymphoid malignancies. Its important to understand that absence of the M-protein does not rule out monoclonal gammopathies as sometimes there is not a detectable concentration within the serum. Sometimes this can lead to a false-negative result. More sensitive tests such as a serum immunofixation test should be performed. Multiple myeloma is a monoclonal increase in IgG immunoglobulins. Other monoclonal gammopathies include waldenstrom macroglobulinemia which is an increase in IgM immunoglobulins. Some more common ones are Al amyloidosis and monoclonal gammopathy of undetermined significance (MGUS).

Polyclonal gammopathies are infectious or various inflammatory processes characterized by a broad-based peak in the gamma fraction. This typically represents a polyclonal immunoglobulin increase seen in autoimmune disease, liver diseases, viral/bacterial infections, and various other malignancies.

Thalassemias

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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.