SHALENDER BHASIN, MD

• Chief and Professor, Department of Endocrinology,
• Diabetes, & Nutrition, Boston University, Boston

Patients whose bleeding is controlled can be discharged and instructed not to take anything by mouth for 4 hours and then only liquids and soft foods diabetes mellitus type 2 treatment guidelines buy genuine duetact. This condition usually occurs when the clot that is normally present in the socket after a tooth extraction becomes dislodged or dissolves diabetes mellitus hyperglycemia purchase 17 mg duetact with visa. The examination is essentially unremarkable with the exception of a missing clot where the tooth was extracted blood glucose 56 cheap 17 mg duetact visa. In only a small percentage of patients (2% to 5%) will a dry socket develop; however early warning signs diabetes type 2 order generic duetact, this number increases with traumatic extractions or impacted third molars diabetic eye problems duetact 17mg without prescription. The pain associated with a dry socket is extremely severe diabetes insipidus vs type 1 purchase duetact 17 mg mastercard, and if a patient is seen several days after an extraction with relatively normal findings on examination and severe pain, it is probably a dry socket. It must be distinguished from osteomyelitis, which is characterized by fever, leukocytosis, malaise, and nausea. The pain related to a dry socket will not be relieved with traditional pain medications, but a dental block usually provides instant relief. Gauze (1 4 inch) impregnated with eugenol (oil of cloves) or a local anesthetic may be used. The socket may also be packed with a slurry of Gelfoam (Pharmacia and upjohn Company) and eugenol. Others are associated with the attachment structures of the teeth such as the gingiva, periodontal ligament, and alveolar bone. These infections are often chronic conditions, but they can progress to the point where periodontal abscesses form and emergency treatment is required. Emergency clinicians will be called on to drain abscesses of dental origin that do not extend into the deep spaces and that have well-defined boundaries easily accessible by intraoral or external drainage. Disease of the Pulp Disease of the pulp can occur as a result of trauma, operations, or other unknown causes, but the most frequent cause is invasion of microorganisms after carious destruction of the enamel. As the enamel is destroyed, caries progresses more rapidly through the dentin and into the pulp chamber and causes an inflammatory response referred to as pulpitis. If the path of carious destruction through the tooth is adequate for drainage of the developing inflammation, the patient may be only mildly symptomatic or even asymptomatic for a long time. If drainage is blocked, however, the process may progress to rapidly involve the entire pulp cavity and the periapical space. Abscesses in the periapical region are generally picked up on dental x-rays and less commonly on a Panorex film. However, unless extension through the cortex exists, it is not important for the emergency clinician to make the distinction between pulpitis and a periapical abscess. A periapical abscess will follow the path of least tissue resistance if not treated. This may be through alveolar bone and the gingiva and into the mouth or into the deep structures of the neck. If the infection has progressed apically through alveolar bone and localized swelling and tenderness are present, incision and drainage should be performed (discussed subsequently). In the absence of trauma or recent instrumentation, it is prudent to begin antibiotic coverage for the typical oral flora. In most cases, perform a supraperiosteal injection (tooth block) with a long-acting anesthetic such as bupivacaine because this not only provides immediate and long-lasting relief but also decreases the requirement for narcotic analgesics once the anesthetic effect has dissipated. Avoid performing a supraperiosteal injection if the abscess has extended through gingival tissue and is present near the injection site. In this case a regional block away from the infected tissue may be more appropriate. If pericoronal infection is localized, local or nerve block anesthesia is followed by removal of submucosal debris. Saline rinses and oral antibiotics are prescribed along with dental follow-up in 24 to 48 hours. Drainage of Dentoalveolar Infections the important determination for the emergency clinician to make is whether the odontogenic infection is localized, confined, and easily accessible or whether it is complex and involves several potential spaces. Likewise, determine whether the patient appears to be in a toxic state, has trismus, or exhibits any signs of airway compromise. Dental infections are not always obvious and can be mistaken for sinusitis, or vice versa. Benzocaine 20% gel, 5% lidocaine gel, or a combination of lidocaine, prilocaine, and tetracaine generally provide good topical anesthesia before injection. Application of these medications to the dry mucosa before injection of the local anesthetic decreases the pain associated with injection. After applying the topical anesthetic, slowly infiltrate local anesthetic with a vasoconstrictor until the tissue blanches. Regional or dental blocks may be performed instead of local infiltration if needle placement would track already infected tissues into healthy areas. Instruments necessary for drainage of dentoalveolar abscesses are those usually found on a standard incision and drainage tray and include hemostats, scalpel (No. Intraoral Technique Intraoral abscesses do not routinely require any antiseptic mucosa preparation before drainage. If the wound is large enough to place a drain or gauze inside, tack one end to the mucosa with a silk suture to prevent aspiration. Advise the patient to perform saltwater rinses hourly and arrange follow-up in 24 to 48 hours with a dentist or oral surgeon to remove the drain and provide continued management. Because the source of the abscess is not always known to the emergency clinician, prescribe antibiotics. Extraoral Technique Most simple dental infections can be drained intraorally, but occasionally, an abscess spreads to the face and requires drainage through the skin. It is important to realize that most dental infections should be drained through the mouth, if possible, because any extraoral drainage will cause some scarring. Never make any incisions on the face in direct proximity to important structures such as the facial nerve or the parotid gland and Disease of the Periodontium Periodontal disease is also very common and affects practically all adults to some degree. Periodontal disease refers to infection of the attachment apparatus of the teeth: the gingiva, periodontal ligament, and alveolar bone. In advanced disease, the gingiva becomes red and inflamed and tends to bleed easily. With chronic periodontal disease, an abscess can form when organisms become trapped in the periodontal pocket. The purulent material usually escapes through the gingival sulcus; however, it occasionally invades the supporting tissues, the alveolar bone, and the periodontal ligament (periodontitis). Antibiotics should be reserved for severe cases or for abscesses that cannot be drained. If it is uncertain whether the abscess originates from the pulp or the periodontium, prescribe antibiotics even if the abscess has been drained. The gingiva overlying the crown may entrap bacteria and debris, and infection may subsequently develop. Typical signs of inflammation and infection may develop, including erythema, edema, pus, and foul breath. Examination of the overlying gingiva with a tongue blade or finger will elicit tenderness and may produce drainage from the infection underlying the tissue flap. The pain may be moderate to severe, and referral of the pain to an ear region is common. The localized infection occasionally spreads to deeper areas such as the pterygomandibular or submasseteric spaces. Clinically, patients with significant spread of a pericoronal infection will have trismus secondary to irritation of the masseter and pterygoid muscles. B, Intraoral examination revealed a pea-sized pointing abscess (arrow) at the base of an upper tooth, the cause of the sinusitis. C, A hemostat was inserted into the abscess cavity and spread open to yield more pus. Intravenous antibiotics (clindamycin) were administered followed by oral antibiotics, and the patient saw her dentist the next day. A, this patient had a chronic toothache for months and got minimally better with antibiotics from numerous emergency departments. B, A Panorex radiograph revealed nonunion of a fractured mandible with osteomyelitis. C, In this case a fractured mandible with osteomyelitis produced an obvious abscess. She had been treated for a presumed dental abscess many times in the past and got temporary relief. Remember that not all infections about the mouth are the result of a simple dental infection. Osteomyelitis of the mandible, various tumors, and other exotic diseases can simulate a dental infection. Deep Space Infections of the Head and Neck It is not unusual for odontogenic infections to spread into the various potential spaces of the face and neck. The signs and symptoms consist of fever, chills, pain, difficulty with speech or swallowing, and trismus. Although infections of certain teeth usually spread to particular contiguous spaces, the rapid spread of these infections often makes localizing the exact space difficult. Any space, including the buccal, temporal, submasseteric, sublingual, submandibular, parapharyngeal, and others, may be involved. Maxillary extension of periapical abscesses can spread into the infraorbital space and, subsequently, to the cavernous sinus through the ophthalmic veins and result in cavernous sinus thrombosis. Cavernous sinus involvement is associated with periorbital cellulitis, as well as meningeal signs or a decreased level of consciousness. The source is usually a decayed lower tooth, with the tooth itself being relatively asymptomatic. As the infection progresses, the submandibular, submental, and sublingual spaces all become edematous, and there may be elevation of the tongue and the soft tissues of the mouth. The suprahyoid region of the neck appears tense and indurated, and landmarks may be obscured. This rapidly progressive disease can cause unexpected or rapid-onset airway compromise, and surgical intervention is warranted, as is consultation with the team that handles difficult airways. Perform airway interventions early if there is any question of compromise, and consider tracheostomy. Administer antibiotics to slow spread of the infection and decrease hematogenous dissemination. The bacteria involved are typically polymicrobial, involving both aerobic and anaerobic bacteria from the primary source. Poverty, homelessness, alcoholism, and psychiatric illnesses are commonly associated conditions. Ethanol intoxication is the most common cause of excessive heat loss in urban settings. Before describing procedures and making recommendations, essential terms are defined and the pathophysiology of hypothermia is briefly reviewed. Hypothermia is not only a common diagnosis in rural areas but has also become more commonplace in urban centers across the nation secondary to inadequate housing or lack of preparation for cold weather changes. The higher the environmental stress, the greater the potential for failure in performance and the development of hypothermia. Everest in 1996, 2014 and 2015, are reminders that even well-protected, acclimatized individuals can succumb to cold-related fatalities. Neonates are at particular risk of hypothermia due to large surface area-to-mass ratio, deficient subcutaneous tissue and inefficient shivering. Acute neonatal hypothermia is common after emergency delivery or resuscitation and has been reported in cases of infant abdandonment. It is a well-accepted practice to carry out resuscitation of these individuals for extended periods. In the majority of studies of urban hypothermia, death has been attributed to the severity of the underlying disease. A common error is failure to routinely obtain an accurate core temperature in all patients at risk. The diagnosis is frequently delayed because of false reliance on standard oral temperatures. Symptoms such as confusion in the elderly and combativeness in intoxicated patients might not initially be recognized as symptoms of hypothermia. Hypothermic patients frequently will not feel cold or shiver, particularly the elderly population, who have impaired thermoregulatory responses because of their advanced age. It occurs as a result of constricted blood vessels near the surface of the body that suddenly dilate. In many cases these patients are mislabeled as psychotic, thereby leading to further delays in appropriate treatment. It is of paramount importance not only for confirmation of the diagnosis but also for guidance in further diagnostic and therapeutic decisions. Any thermometer that does not record temperatures in the hypothermic range is inappropriate for evaluating significant hypothermia. An electronic probe with accompanying calibrated thermometer is recommended when monitoring this vital sign.

It is usually possible to diagnose a septal hematoma by inspecting the nasal septum with a speculum for swelling diabetes 400 reading generic 17 mg duetact visa, pain diabetes symptoms webmd quality 16mg duetact, and a fluctuant area diabetes insipidus without thirst generic 17 mg duetact otc. The presence of septal asymmetry with a bluish or reddish hue of the mucosa is suggestive of a septal hematoma diabetes glucose levels purchase generic duetact pills. Direct palpation with the littlest finger may be necessary because newly formed hematomas may not yet be ecchymotic diabetes type 1 erectile dysfunction safe duetact 16 mg. Palpation can further differentiate septal hematoma from septal deviation diabetes diet oats cheap 16 mg duetact with amex, which may appear to be similar because of asymmetry. The best way to palpate for a septal hematoma is to insert the gloved small fingers in each side of the nose and palpate the entire septum to feel for swelling, fluctuance, or widening of the septal space. Caution should be used in those with known bleeding diathesis or those who are taking anticoagulants. Use small cup forceps or scissors to remove enough mucosa to prevent premature closure of the wound and reaccumulation of the hematoma. Procedure Treatment of a septal hematoma consists of evacuation of the clot with subsequent reapproximation of the perichondrium to the cartilage. To drain the hematoma, incise the mucosa over the hematoma horizontally after adequate anesthesia is achieved. Excise a small amount of mucosa to prevent premature closure of the incision and place a section of a sterile rubber band to act as a drain. Pack the nostril, as for anterior epistaxis, to reapproximate the perichondrium to the cartilage. When there is no further hematoma formation over a 24-hour period, remove the drain. Pack the affected naris for one more day to complete the apposition of perichondrium to cartilage where the drain had been. Complications Though rare, nasal septal abscess formation is the most common complication of septal hematomas. The infection can spread to the sinus cavities and lead to meningitis, cavernous sinus thrombosis, intracranial abscess, and orbital cellulitis. A large or rapidly expanding hematoma may cause pressure on the septum and lead to avascular necrosis of the septal cartilage. The nasal septum can collapse and lose its shape, which causes a noticeable cosmetic defect. After drainage, the hematoma may recur and should be treated by repeated drainage to prevent cartilage damage. Reaccumulation can be prevented by incising a piece of mucosa before packing the nasal cavity. Nasal fractures are accompanied by a broad range of symptoms, including mild swelling, epistaxis, and periorbital ecchymosis with obvious deformity. As with any trauma involving the head, evaluate for coexistent intracranial injury or neck injury. In the evaluation of nasal trauma, rule out the existence of a septal hematoma or cerebrospinal fluid rhinorrhea. In most cases the swelling and soft tissue deformity prevent adequate evaluation, treatment, or both. Evaluation of a patient with a suspected nasal fracture includes a thorough history, external nasal examination, and internal nasal examination using a nasal speculum with or without the use of a rigid nasal endoscope. Nasal radiographs are not routinely needed because they will not alter the course of treatment or injury. Refer the patient to an otolaryngologist or plastic surgeon for reexamination and definitive treatment in 3 to 5 days. Stress the importance of reevaluation within 10 days so that the bones do not set in a misaligned state. Indications and Contraindications the indications for reduction of a nasal fracture are first based on the type of deformity and degree of swelling present (timing of reduction). Other indications include less than 3 hours after injury in adults and children if minimal edema is present, reduction 6 to 10 days after injury in adults once the edema has resolved but before setting of the fracture fragments, and reduction 3 to 7 days after injury in children once the edema has resolved. Equipment the standard equipment of nasal decongestant and anesthetic are needed; in addition, nasal speculum, bayonet forceps, Frazier suction tip, anterior nasal packing material, a good light source, and some specialized equipment are needed, including elevators (goldman, Boies, Salinger, Ballenger), Walsham forceps for grasping the nasal bones, and Asch forceps for reduction of the septum. Procedure Most fractures and patients with significant soft tissue swelling should be seen in follow-up for definitive evaluation and possible reduction of the fracture. Some patients prefer immediate correction, are not concerned with aesthetics, or are unable to comply with Nasal Fracture Reduction 1 Unilateral fracture of the nasal pyramid 2 Dislocation of the septum Nondisplaced and minimally displaced nasal fractures often do not require manipulation, but the true extent of the deformity is difficult to appreciate initially. Reduction of a depressed and dislocated nasal bone fracture is usually performed in 3 to 7 days, after the swelling has subsided and the true deformity is obvious. After marking the distance of the intercanthal line on the elevator with a thumb, the tip of the instrument is used to reduce the medialized fragment by elevating it. The opposite thumb may simultaneously reduce a contralateral outfractured nasal bone (pyramid). Use upward and outward force, perpendicular to the plane of the dorsum, to lift the septum until it is no longer overlapping. To minimize potential litigation, obtain written consent and take prereduction and postreduction photographs. Inform the patient that the outcome is not guaranteed because impacted fractures may not reduce and greenstick fractures may deform again after reduction. A specific nasal elevator (Boies or Joker) or the handle of a metal scalpel can be used. The depth of insertion is determined by placing the instrument against the surface of the skin on the lateral aspect of the nose, with the distal tip at the intercanthal line. Mark the position of the instrument at the alar rim, insert the instrument into the nose, and stop 1 cm short of the measured depth. Lift the elevator anteriorly and laterally until the depressed fragment is in proper position. If reduction with the elevator is unsuccessful, use Walsham forceps to manipulate the nasal bones. Insert one arm of the forceps into the nasal cavity and the other against the surface of the skin. Firmly grasp the displaced bone, disimpact, and reposition it into the correct location. Insert each arm of the instrument on either side of the nasal septum and position it. Use an upward and outward force perpendicular to the plane of the dorsum to lift the septum until it is no longer overlapping. After the maneuvers, assess the reduction for proper alignment or subsequent displacement secondary to a greenstick fracture. If either occurs, refer the patient to an otolaryngologist to see whether open reduction is necessary. Stabilize the reduction internally with nasal packing and externally with an exterior splint dressing (Thermaplast or Aquaplast). Some authors believe that splinting will mask an incomplete reduction or adversely manipulate the reduction during placement. Reduction may produce a nasal septal hematoma, which if untreated, can lead to cartilage destruction and deformity. Direct infiltration of anesthetic may result in nerve damage, dysesthesias, or paresthesias after the effects of the anesthetic wear off. Patients may also have unilateral purulent or bloody nasal discharge, unilateral sinusitis, or recurrent unilateral epistaxis. Injuries can occur and include mucosal burns, ulcerations, liquefaction necrosis, septal perforation, synechiae, and stenosis of the nasal cavity. These small, commercially available earth magnets are usually worn on either side of the alar cartilage and give the appearance of a pierced nasal stud. The magnetic attraction can be quite strong and may lead to pressure necrosis of the nasal mucosa and possibly septal perforation. This attraction can also make removal difficult, as well as painful for the patient. Metallic or calcified objects may show up on radiographs, but physical examination remains the most reliable means for diagnosis. Although admission incurs increased cost, has inherent procedural risks, and causes psychological stress in parents and patients, providers should be prepared to call a consultant after several attempts. Repeated attempts may result in trauma and displacement of the object into a less favorable location. Do not attempt mechanical removal if the object appears to be out of range of the instruments. In addition, do not attempt removal in an uncooperative patient unless sedation is provided. Use sedation cautiously because it may increase the risk for aspiration, especially when using agents that blunt the protective airway reflexes. Before attempting removal, anesthetize and vasoconstrict the mucosa of the affected naris topically. Place more cooperative patients in the sniffing position and use a headlamp for proper illumination. Local vasoconstriction and anesthesia are helpful, and 2% lidocaine with epinephrine (local anesthetic) may be used. For harder or larger objects, carefully pass a wire loop For an object that cannot be removed with anterior instrumentation, one consideration is to attempt removal with a balloon catheter. Slowly withdraw until resistance is met and then pull the object out of the naris. Attempt to firmly compress the bag as the patient exhales (an assistant is helpful to hold the mask snugly and to occlude the other nostril). Place the patient in the supine position and apply a vasoconstrictor and anesthetic to the nasal mucosa. With a 5-mL syringe attached and the catheter lubricated with lidocaine gel, pass the tip above the object and into the nasopharynx. Inflate the balloon with air or water (2 mL in small children and 3 mL in older children) and control the syringe plunger and balloon size with your thumb. Withdraw the catheter until resistance is felt, and then slowly pull the object out. Complications mentioned in the literature include mild posttraumatic bleeding, as well as the theoretical risk for airway obstruction by the balloon or aspiration from further displacement of the object. The simplest way is to ask patients to blow their nose while occluding the unaffected nostril. Occlude the opposite nostril and apply the Sellick maneuver to prevent passage of air into the esophagus. This technique often requires restraint and can also be threatening to a young child. If successful, the object will move within grasping reach of an instrument or pop completely out of the naris. Instruct the child to make a tight seal, as though drinking, and ask the parent to deliver a quick puff. Use a male-to-male adapter at the end of oxygen tubing to direct the flow of oxygen into the contralateral (nonobstructed) nostril. A, Attach suction tubing to wall oxygen or medical air, and use a suction adapter inserted in the contralateral naris. B, An alternative technique uses oxygen tubing, an earpiece from a disposable stethoscope, and an 8-Fr suction catheter. Two other techniques using positive pressure have been described and use simple equipment and oxygen or medical air. In the first technique,63 wall oxygen or medical air, suction tubing, and a suction-tubing adapter are needed. Attach one end of standard suction tubing to the wall oxygen or compressed air unit. The plastic suction-tubing adapter should be firmly placed into the opposite end of the suction tubing. C, this patient was suspected of having a dystonic reaction because she could not speak and her mandible was misaligned. Titrate the oxygen to high flow (usually 10 to 15 L/min) until the foreign object is expelled. The other technique uses a disposable stethoscope earpiece, 8-Fr suction catheter or feeding tube, oxygen, and oxygen tubing64. Connect the device to the oxygen source with tubing and set the oxygen to 15 L/min. Next, place the device in the unaffected nostril, release the pressure, and immediately remove the device. If an 8-Fr feeding tube is not available, an 8-Fr suction catheter may be used as an alternative. Bleeding and mucosal laceration are the most commonly reported complications of removal. Inadvertent posterior dislodgement of the object may occur and result in ingestion or aspiration of the object. Spasm of the masseter, internal pterygoid, and temporalis muscles occurs during an attempt to close the mandible. Predisposing factors include anatomic disharmony between the mandibular fossa and the articular eminence and weakness of the capsule and the temporomandibular ligaments.

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Serum Free Light Chains Concentrations of the free serum and light chains can be determined nephelometrically diabetes symptoms hair loss purchase duetact with a mastercard, by using antibodies specific to light chain epitopes that are exposed in free but hidden in heavy-chain bound molecules control diabetes naturally diet order discount duetact on-line. Because of its superior convenience and comparable sensitivity to urine electrophoresis blood sugar up and down 17 mg duetact, this assay is being frequently used in diagnosis diabetes medications for elderly buy cheap duetact 16 mg on-line, prognosis diabetes control chart purchase duetact with american express, and therapeutic response monitoring in light-chain myeloma diabetes diet options cheap duetact 17 mg online, amyloidosis, and smoldering multiple myeloma. Despite being useful for quantitating normal immunoglobulins, they are not accurate when measuring paraproteins. For example, multimer-forming IgA and pentamer-forming IgM paraproteins have a propensity to disassociate into smaller molecules, therefore producing erroneous results. Redissolved cryoprecipitate is then separated on electrophoresis and subjected to immunofixation, which reveals the culprit immunoglobulin. Cryoglobulinemia may due to (1) a monoclonal immunoglobulin, usually IgM, (2) a monoclonal IgM with rheumatoid activity binding to polyclonal IgG ("mixed cryoglobulinemia"), or (3) polyclonal IgM bound to polyclonal IgG. The latter two are associated with a variety of lymphoproliferative or autoimmune disorders and infections, particularly hepatitis C. Elevated serum levels may be found in inflammatory conditions, renal failure (due to impaired excretion), and in lymphoid and plasma cell malignancies, where it is used as an important marker. Serum levels are not useful to follow chemotherapy responsiveness in myeloma because of the lack of specificity but may predict early relapse following autologous transplantation. There are five distinct isoenzymes, based on different ratios of M and H subunits, with identical function but different distribution; distinguishing between these is seldom useful in hematology practice. Elevated serum levels, which can be measured by an automated enzyme activity-based assay, are seen in tissue damage regardless of etiology, as the enzyme is released from necrotic cells into the circulation. In hematology, marked elevations are usually noted with intravascular or intramarrow hemolysis, hemophagocytic lymphohistiocytosis, and tumor lysis syndrome. Serum Uric Acid Uric acid, the end product of purine nucleotide metabolism in humans, is produced in a variety of cells and cleared by the kidneys. Elevated serum levels (hyperuricemia), as measured by an automated enzymatic assay, are seen with either increased uric acid production (increased cell turnover, as in tumor lysis syndrome) or impaired renal clearance (kidney disease and certain medications). Performance evaluation of the latest fully automated hematology analyzers in a large, commercial laboratory setting: a 4way, side-by-side study. Visual inspection of blood films vs automated analysis of red blood cell distribution width. Quantitative anisocytosis as a discriminant between iron deficiency and thalassemia minor. Relationship of reticulocyte age to polychromasia, shift cells, and shift reticulocytes. Automated reticulocyte counting: state of the art and clinical applications in the evaluation of erythropoiesis. Platelet production and platelet destruction: assessing mechanisms of treatment effect in immune thrombocytopenia. Changes in automated complete blood cell count and differential leukocyte count results induced by storage of blood at room temperature. Bone marrow aspiration before bone marrow core biopsy using the same bone marrow biopsy needle: a good or bad practice A bone marrow report of absent stainable iron is not diagnostic of iron deficiency. Single value of serum transferrin receptor is not diagnostic for the absence of iron stores in anaemic patients with rheumatoid arthritis. Hyperferritinemis is associated with insulin resistance and fatty liver in patients without iron overload. Use of a fixed activated partial thromboplastin time ratio to establish a therapeutic range for unfractionated heparin. Unprovoked recurrent venous thrombosis: prediction by D-Dimer and clinical risk factors. A randomized clinical trial comparing, point-of-care platelet function assays and bleeding time in healthy subjects treated with aspirin or clopidogrel. False-negative results in detection of monoclonal proteins by capillary zone electrophoresis: a prospective study. Highly sensitive, automated immunoassay for immunoglobulin free light chains in serum and urine. Long-term biological variation of, serum protein electrophoresis M-spike, urine M-spike, and monoclonal serum free light chain quantification: implications for monitoring monoclonal gammopathies. Braylan Flow cytometry is a technology used routinely in most hematology laboratories. Its entry into the mainstream of clinical laboratory analysis has been aided by the increasing the availability of monoclonal antibodies that define cell surface and intracellular proteins as markers of cell lineage, differentiation, activation, and other biologic properties. Instrument design advances have yielded benchtop cytometers with fixed optics that, when linked with new developments in fluorochrome chemistry, enable a wide range of clinical applications. In addition, proficiency testing is now available in support of these clinical applications through the College of American Pathologists as mandated by the Clinical Laboratory Improvement Amendment of 1988. The major advantage that flow cytometry provides is its capacity to assess multiple measurements on large numbers of individual cells. Flow cytometric studies have extended the understanding of hematopoietic cell development, differentiation, activation, and apoptosis. In addition, they have provided important information regarding hematologic malignancies, insight into reconstitution after stem cell transplantation, and understanding of cell abnormalities that result in immune or hematologic deficiencies. As such, flow cytometry plays an important role in the diagnosis, characterization, and monitoring of a number of hematologic disorders. The basic design of a flow cytometer involves four major elements: Optics, fluidics, electronics, and a computer equipped with specialized software. At the opposite side of the optical bench, light generated from the cells that have intersected the excitation beam is filtered, reflected by dichroic mirrors set in fixed locations, and finally collected by linked photodetectors to allow quantitation of the emitted light at specific wavelengths. To ensure that all cells analyzed experience consistent exposure to the excitation beam, the fluidic system must maintain the cells in a consistent location as they move sequentially through the beam. To accomplish this, the cell suspension is injected into a flowing stream of sheath fluid that hydrodynamically focuses the inner stream of cells within the outer sheath fluid stream. The various light signals (parameters) are collected by the optical bench, while instrument design determines the number of parameters collected per cell. The two reagent-independent (nonfluorescent) parameters are forward-angle light scatter, as a marker of cell size, and side-angle light scatter, as an index of cellular regularity/ granularity. The combination of these two parameters allows for an approximate discrimination among the three major types of leukocytes, as well as evaluation of red blood cells and platelets in whole blood samples. Fluorochromes are excited by light of a defined wavelength and emit light of lower energy (longer wavelength). There are currently different fluorochromes used in clinical flow cytometry, including fluorescein isothiocyanate, phycoerythrin, peridinin chlorophyll protein, and allophycocyanin and many others that can be excited by newly introduced lasers. Combinations of two fluorochromes linked to each other have been developed; they depend on the transfer of energy from the first fluorochrome to excite the second fluorochrome. These tandem fluorochromes extend the range of emission wavelengths available from one excitation beam. The availability of multiple fluorochromes that absorb light of the same wavelength but emit light at different wavelengths means that multiple reagents can be used simultaneously with a single light source to yield a multicolor (polychromatic) study. One or more additional light sources are present in most current clinical instruments to extend the range of multicolor studies. Extended multicolor studies require complex color compensation and data management processes that typically involve sequential data evaluation. The clinical application of flow cytometry in hematology saw its earliest use as a supplement to the morphologic classification of leukemias and lymphomas, affording not only lineage information but also the state of differentiation and/ or maturation,4,5 cell growth, and apoptosis. Fixation and permeabilization facilitate intracellular entry of reagents to determine specific proteins and to assess functional characteristics. Alternatively, the signal intensity of two correlated parameters can be plotted versus cell frequency; the latter displayed as dot density (dot plot. When measuring multiple parameters (as colors), the data are typically evaluated by using dual-parameter displays. Depending on the application, 10,000 to 100,000 events are collected to provide sufficient numbers of cells for meaningful data relative to the subpopulations of interest. This approach applies to well-defined populations with homogeneous antigen expression, but modifications or alternative interpretations may be necessary when the cell populations analyzed are heterogeneous or express dim fluorescence, which can lead to rather arbitrary calculations of percentages of positive cells. The data generated by the computer are only as good as the instrument capabilities, settings, reagents, and cell preparation used. The use of validated reagents is also part of good laboratory practices, while the quality of the cell preparation can be assessed using the nonfluorescent parameters, forward-and sideangle light scatter, to confirm the population of interest. Platelets are obviously smaller than all other blood cells and heterogeneous in size, characteristics that can be confirmed in comparison to red blood cells. Erythrocytes have a characteristic appearance based on forward-and side-angle light scatter. However, because of the large difference in frequency of circulating erythrocytes and leukocytes, there is no practical concern of lymphocytes contaminating erythrocytes (a collection of 10,000 erythrocytes normally include less than 20 lymphocytes). In part because of this reason, the study of lymphocytes in a whole blood sample or other specimens containing a significant amount of blood typically involves a red blood cell lysis step to eliminate erythrocytes. Following successful red cell lysis, a three-part differential is observed in peripheral blood with normal lymphocytes representing the smallest (forward-angle light scatter) and most regular/ agranular (side-angle scatter) cells, while granulocytes are slightly larger (higher forward-angle scatter) and show substantial granularity (high side-angle scatter), and monocytes fall between these two cell types. The two less prevalent granulocyte types differ in their location on a scatter plot, with eosinophils typically falling within the granulocyte population while basophils overlap with lymphocytes. Hematopoietic stem cells are normally found in the lymphocyte area of the scatter plot. It is important to recognize that the cell relationships noted previously do not necessarily apply in cases of hematopoietic malignancy because neoplastic cells may exhibit altered light scatter properties or appear as a distinct population, separate from normal elements. The interpretation of fluorescent data based on antibody binding reflects the biology of the particular cognate cell surface protein. In both histograms, there are at least three cell populations: Cells negative for the marker, cells showing intermediate fluorescence, and cells that have bright fluorescence. Many monoclonal antibodies, individually or in combination, can serve to distinguish cells of a specific lineage (Table 28. As mentioned earlier, nonfluorescent parameters of forward-angle and side-angle scatter help distinguish among lymphocytes, monocytes, granulocytes, and platelets. Within the lymphocyte population, lineage-specific antibodies differentiate various populations and subpopulations. A combination of additional antibodies often clarifies the relative expression of different antigens on specific cell populations. Cell surface proteins may be altered under different circumstances during the life cycle of a cell, including preferential expression early and/ or late during differentiation, expression in response to cell activation, and/ or in various states of cell-specific function. Protein upregulation implies a range of expression that could include cells transforming from negative to clearly positive, depending on the temporal pattern of expression. In some circumstances, isoforms of a specific protein are differentially expressed, and cells may express one or the other isoform or both. The histogram overlay demonstrates that there is a shift in the stained cells that would not be adequately reflected by simply scoring cells as positive or negative. These considerations are particularly relevant for many markers used to evaluate malignant cells. In fact, consensus groups have repeatedly emphasized that reporting percentage values when interpreting results in hematopoietic malignancies is generally unsatisfactory. For this reason, it is recommended that interpretation of flow cytometric results in hematopoietic and lymphoid malignancies be based on the visual examination of the plots for each of the antibodies used, and that the results be primarily descriptive, in a manner similar to the microscopic evaluation of cells or tissues. Numerical values are only used to indicate the fraction of neoplastic cells or other well-defined cell populations present in the sample. Flow cytometry has also been applied to investigate intracellular characteristics and, specifically, proteins that may only be detected intracellularly13 or as well as those that ultimately are also expressed on the cell surface. Flow cytometry has revolutionized the manner in which we diagnose, classify, and monitor acute leukemia or lymphoproliferative disorders, and it is rare now that patients with these disorders are treated without including the flow cytometric data. The technology is rapid, quantitative, and can analyze simultaneously multiple antigens in a large number of cells, allowing the easy detection, characterization, and enumeration of malignant cells, even when admixed with normal elements. The ability of flow cytometry to recognize malignancy is based on its capacity to distinguish differences in antigen expression between normal and neoplastic cells. Normal hematopoietic cells originate from a stem cell in the marrow that subsequently gives rise to a progeny of different cell lineages. These cell progenitors traverse various developmental stages and ultimately evolve into mature elements in the circulation and other peripheral organs. As the hematopoietic cells develop and differentiate, they undergo changes in their surface or intracellular antigenic profile that is characteristic of their lineage and differentiation stage. Hematopoietic malignancies are clonal cell populations that express similar antigens to those of their nonneoplastic counterparts but usually with a different expression pattern that is unique for each type of neoplasia. This antigen expression can be increased, decreased, absent, asynchronous, or may be of a different cell lineage. Thus, knowledge of the immunophenotype of a cell together with its physical properties revealed by light scatter signals allows determination of not only their lineage and developmental stage but also, in most instances, their normal or neoplastic nature. Furthermore, in T-or B-cell lymphoproliferative disorders, the clonal nature of the lymphocytes can be established by recognizing the restriction of expression of immunoglobulin light chains28 or T-cell receptor beta chains. This identification is possible due to the antigenic expression and/ or physical properties of neoplastic cells that are different from those of their normal counterparts. Although genetic abnormalities in acute leukemic cells can be recognized by molecular genetic techniques, flow cytometry is an excellent alternative in cases where genetic markers are absent. Many applications in nonmalignant diseases are also routinely performed in the clinical laboratory.

Genetic counseling is complicated by the occurrence of patients with normal development [18 treatment juvenile diabetes purchase duetact line, 29] blood sugar vomiting discount duetact 17mg on line. The biochemical hallmark of these diseases is the accumulation of D-2-hydroxyglutaric acid in body fluids diabetes type 2 and fatigue buy 16 mg duetact overnight delivery. The compound is readily detected and quantified by organic acid analysis of the urine diabetes type 1 treatment new purchase duetact uk. The chiral center at the asymmetric second carbon results in differential rotation of polarized light shone on the two compounds diabetes test kit in india buy duetact 17 mg. Light is rotated to the right by the D- (Latin dexter) form and to the left by the L- (Latin laevus) compound diabetes signs in 1 year old generic duetact 17 mg without a prescription. Urinary excretion of D-2-hydroxyglutaric acid ranged from 18 (this patient also recorded a level of 1072) to 7076 mmol/mol creatinine [1, 2, 5]. One patient with severe disease had intermittently normal and high levels of excretion [13]. Excretion of 2-oxoglutaric acid ranged from 404 to 862 mmol/mol creatinine [1], amounts similar to those reported in 2-oxoglutaric aciduria [32]. This was found in other patients [5], and other citric acid cycle compounds were up in some, usually to a lesser level [5]. Decreased levels of carnitine have been found in many patients, but many have been receiving valproate [5]. Acylcarnitine profiles have been normal, but one had multiple elevations, as seen in multiple acylCoA dehydrogenase deficiency, but without excretion of glutaric and ethylmalonic acids. Increased levels of lactic acid in the urine were found in a few patients [5, 13]. In studies of cultured fibroblasts [29, 33], the media in which cells derived from patients with D-2hydroxyglutaric aciduria grew contained 5 to 30 times the control concentration of D-2-hydroxyglutaric acid. Studies of cultured human lymphoblasts incubated with 13 C-labeled glucose or 3H-labeled glutamate indicated that D-2-hydroxyglutaric acid is rapidly converted to 2-oxoglutaric acid [34]. Mean activities in control fibroblast and lymphoblast homogenates were 208 + 207 and 1670 + 940 pmol/hour/mg protein. The reaction is also catalyzed by a transhydrogenase and in the exchange 4-hydroxybutyric acid is converted to succinic semialdehyde [36]. The enzyme is highly active in the liver and kidney, but it is also active in the brain and heart. Two patients with severe disease were found to have mutations in the dehydrogenase gene [7]. The intronic mutation created an alternative spliceacceptor site leading to a frameshift and premature stop. Mutations were also found in two unrelated consanguineous Palestinian families; in one of which, two affected siblings were asymptomatic; in the other, the patient had mild disease consisting of absence seizures readily controlled, difficulty reading, hyperactivity, and behavior problems [9]. In the other family, the patient was homozygous for an A to G transition at nucleotide 1315 which changed asparagine 439 to aspartic acid. The duplication resulted in a frameshift which substituted anginine by glutamic acid at 110 and termination of the 19th codon. The unusual differences in the phenotype make genotype/phenotype correlations problematic. In the most recent [10] assessment, 29 mutations were found, 21 novel, each of which predicted a truncated protein. Molecular genetic abnormalities were found in all patients in whom deficiency of enzyme activity was documented. The key to the molecular defect in type 2 patients came from observations in cancer cells in which D-2hydroxyglutarate accumulated in glioblastoma cells with superactivity of isocitrate dehydrogenase. This mutation interferes with the normal ability of the enzyme to convert isocitrate to 2-ketoglutarate and confers a new function in the conversion of 2-ketoglutarate to D-2-hydroxyglutarate. In 15 patients, heterozygous G to A substitution at position 419 replaced the arginine at 140 with glutamine (p. These gains of function mutations confer an additional enzymatic capacity to convert 2-oxoglutarate to D-2-hydroxyglutarate. The greater amounts of D-2-hydroxyglutarate in type 2 than in type 1 are explained by the superactivity which overwhelms the activity of the normal dehydrogenase for D-2-hydroxyglutarate. In eight of nine sets of parents, the mutation could not be detected, indicating a denovo mutation and establishing the disease as a dominant. In one family, three affected offspring of a mother with normal excretion of D-2-hydroxyglutarate suggested germ line mosaicism. Deficiency impairs the efflux of citrate and isocitrate from mitochondria in exchange for malate. This leads to depletion of cytosolic citrate and accumulation of mitochondrial citrate. A variety of mutations was found in 12 patients, eight of whom were homozygous [12]. The product 4-hydrox ybut y ric acid is a lso neuropharmacologically active, as illustrated by patients with 4-hydroxybutyric aciduria which is due to succinic semialdehyde dehydrogenase deficiency (Chapter 13). Fibroblasts derived from patients with D-2-hydroxyglutaric aciduria have been found to have normal transhydrogenase activity; on the other hand, it is likely that this enzyme is responsible for the occurrence of D-2-hydroxyglutaric aciduria in patients with 4-hydroxybutyric aciduria (Chapter 13). In patients with multiple acylCoA dehydrogenase deficiency (glutaric aciduria type 2) [39] (Chapter 45), 2-hydroxyglutaric acid excretion is elevated, and it is the D-isomer that is predominant. Of course, in glutaric aciduria type 2, any hydroxyl acid accumulated might lead to the formation of D-2-hydroxyglutaric aciduria in the presence of 2-oxoglutarate in a transhydrogenase reaction. The pathophysiology of the disease is thought to represent a developmental neurotoxicity of D-2hydrox yglutaric acid. Incubation of pathologic concentrations of the compound with primary neuronal cultures from chickens and rats led to excitotoxic cell damage via activation of the N-methyl-D-aspartic acid receptor [40]. D-2-Hydroxyglutaric acid inhibited creatine kinase [11] in brain and in skeletal and cardiac muscle, and cytochrome c oxidase activity in fibroblasts in vitro, but electron transport chain activity in the fibroblasts of patients was normal. D-2-hydroxyglutaric acid was also found to inhibit in vitro the activity of cytochrome oxidase in rat brain fractions [11]. Patients with combined D-2 and L-2-hydroxyglutaric aciduria urine excrete increased quantities of citric acid cycle intermediates, 2-ketoglutarate, malate, fumarate and succinate. In the dysmorphic siblings [28, 29], D- and L-2hydroxyglutaric acids were elevated (D-449 mmol/mol creatinine and (L-46), although on one occurrence neither were elevated in patient two; so, the diagnosis can be missed with only one assay. Elevated lactate, global hypotonia and encephalomyopathy are the clinical features found broadly in patients with mitochondrial disease. Pro45Leu) were reported in siblings who had prominent facial dysmorphic features and lactic acidosis [28]. On the other hand, establishment of the molecular nature of the gene and enzyme represent major additions to the understanding of this disease. Inhibitors of the superactive enzyme in the type 2 disease are under clinical exploration. In the combined disorder, evidence of depletion of cytosolic citrate and accumulation of citrate within mitochondria led to treatment with malate which was ineffective; in contrast, treatment with citrate (1500 mg per Kg) led to increased urinary malate and succinate and remarkable control of seizures [42]. D-2hydroxyglutaric aciduria in a newborn with neurologic abnormalities: a new metabolic disorder D-2-hydroxyglutaric aciduria in a neonate with seizures and central nervous system dysfunction. D-2-hydroxyglutaric aciduria and glutaric aciduria type 1 in siblings: coincidence, or linked disorders Mutations in the D-2-hydroxyglutarate dehydrogenase gene cause D-2hydroxyglutaric aciduria. Metaphyseal enchondrodysplasia with 2-hydroxyglutaric aciduria: observation of a third case and further delineation. Spondyloenchondromatosis with D-2-hydroxyglutaric aciduria; a report of a second patient with this unusual combination. D-2-hydroxyglutaric aciduria: a case with an intermediate phenotype and prenatal diagnosis of two affected fetuses. D-2hydroxyglutaric aciduria and glutaric aciduria type 1 in siblings: coincidence, or linked disorders D-2-hydroxyglutaric aciduria with absence of corpus callosum and neonatal intracranial haemorrhage. D-2-hydroxyglutaric aciduria: Hypotonia cortical blindness seizures cardiomyopathy and cylindrical spirals in skeletal muscle. Metaphyseal enchondrodysplasia with 2-hydroxy-glutaric aciduria: observation of a third case and further delineation. Combined D-2 and L-2-hydroxyglutaric aciduria with neonatal onset encephalopathy: a third biochemical variant of 2-hydroxyglutaric aciduria Stable isotope dilution analysis of d- and L-2-hydroxyglutaric acid: application to the detection and prenatal diagnosis of d- and L-2hydroxyglutaric aciduria. Chiral liquid chromatography tandem mass spectrometry in the determination of the configuration of 2-hydroxyglutaric acid in urine. Disease-related metabolites in culture medium of fibroblasts from patients with D-2-hydroxyglutaric aciduria L-2-hydroxyglutaric aciduria and combined D/L-2-hydroxyglutaric aciduria. Investigations by mass isotopomer analysis of the formation of D-2-hydroxyglutarate by cultured lymphoblasts from two patients with D-2-hydroxyglutaric aciduria. Measurement of D-2-hydroxyglutarate dehydrogenase activity in cell homogenates derived from D-2-hydroxyglutaric aciduria patients. Isolation and characterization of a hydroxyl-acid-oxoacid transhydrogenase from rat kidney mitochondria. Demonstration of 4-aminobutyric acid aminotransferase deficiency in lymphocytes and lymphoblasts. Inhibition of cytochrome coxidase activity in rat cerebral cortex and human skeletal muscle by D-2-hydroxyglutaric acid in vitro. Molecular phylogeny of sequenced Saccharomycetes reveals polyphyly of the alternative yeast codon usage. A survey of eight patients, in 1992 [2], and a later report by Barth and colleagues [3], established the usual phenotype of slowly progressive mental impairment and cerebellar signs with onset in late infancy. Mutations in the gene were found independently by these authors and by Rzem et al. About 100 different mutations have been identified [7], many of which lead to a truncated protein. The enzyme acts as a metabolite repair enzyme [6] which catalyzes the conversion of L-2-hydroxyglutarate formed in the malate dehydrogenase reaction back to 2-oxoglutarate. Delay in walking, abnormal gait, delay in speech, muscular hypotonia, and febrile seizures have been the presenting complaints in seven of 12 patients [3]. Progressive ataxia, variable extrapyramidal and pyramidal signs, epilepsy, and progressive mental retardation develop. In four patients, learning disability in school first called attention to the disease. In one, cerebellar signs at ten years of age were the first evidence of disease recognized. We reported [9] a patient with a much more severe phenotype who presented with disease that was rapidly fatal by 28 days of life. Cerebellar manifestations were prominent in all but one of the patients summarized by Barth et al. Progressive deterioration was documented in one patient [8] after a number of years of relative stability; by 16 years, she was unable to walk and had repeated seizures. Progression was also reported in two patients by Divry and colleagues [10], who emphasized ataxia, brisk tendon reflexes, and positive Babinski as extrapyramidal signs and first reported macrocephaly in both patients. Two adult Japanese patients had seizures in childhood and psychomotor impairment, but began progressive degeneration after 25 years of age [12]. Another 15-year-old boy [13] was wheelchairbound and had impaired mental development epilepsy, optic atrophy, spastic tetraparesis, and dystonia. A very different clinical picture was exemplified by a female patient who was limp at birth and had poor respiratory effort and bradycardia [9]. Initial pO2 was 18, and Apgar scores at 1, 5, and 10 minutes were 3, 6, and 8, respectively. At 80 minutes, there was profound apnea and cyanosis requiring assisted ventilation. At two years of age, she developed grand mal seizures and progressive ataxia became evident. Thereafter, the clinical picture remained stable until adolescence, when epilepsy reoccurred, and she lost the ability to walk [8]. Please note signal changes in the globi pallidi (Reproduced with permission from [15]). The pattern on neuroimaging has been stated to be unique among neurodegenerative disorders [3]. A brain tumor (an ependymoma) was found in a 17-yearold boy with L-2-hydroxyglutaric aciduria, and seven other patients have been identified with brain tumors, suggesting an increased risk [18, 19]. The observed malignancies varied in type and included astrocytoma, primitive neuroectodermal tumors, medulloblastoma, and oligodendroglioma. Diffuse gliomatosis cerebri affecting both cerebral hemispheres and upper brainstem was reported in an adult [19]. There was striking astrocytosis of the white matter in an olivopontocerebellar distribution. In a 15-year-old boy [20], cortical neuronal loss was accompanied by intense gliosis and spongiosis and vacuolation in the subcortical white similar to that found in Canavan disease. Many families have had more than one affected offspring, and males and females have been similarly affected. It is essential to determine the optical configuration of the compound identified because D-2-hydroxyglutaric aciduria (Chapter 11) is a different disease. In the cerebellum, folial atrophy involved the vermis particularly, and there were signal changes in the dentate nuclei. Magnetic 98 L-2-hydroxyglutaric aciduria Prenatal diagnosis is feasible using this method. Abnormalities in concentrations of carnitine or dicarboxylic acids have not been found [3]. L-2-Hydroxyglutarate was not a known intermediate in any eukaryotic metabolic pathway. Ion exchange chromatography separated two enzymes, D-2hydroxyglutarate dehydrogenase and L-2-hydroxyglutarate dehydrogenase.