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Specimens Information and Courses from MediaLab, Inc.

These are the MediaLab courses that cover Specimens and links to relevant pages within the course.

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Basic Tissue Orientation and Paraffin Embedding Technique
Introduction

The focus of this course will be on the technical process of producing human tissue blocks embedded in paraffin wax. Other embedding techniques using various media such as gelatin, ester wax, polyethylene glycol, and epoxy resin are also used in some histological techniques. However, in this course we will be discussing the method of embedding human tissue samples in molten, or melted, paraffin wax. This is the most commonly utilized method for routine tissue embedding and is the method most utilized in nearly all hospital histopathology laboratories for processing human tissue samples for diagnostic interpretation.In the order of events (chronology) of the total histology process, paraffin embedding takes place following tissue processing and prior to and in preparation for microtomy. For proficiency in paraffin embedding, the histologist needs:An understanding of basic anatomy for use in tissue type orientationKnowledge of basic tissue sampling methods used in gross dissectionTo develop manual dexterity and spatial reasoning in order to correctly orient the specimen in the tissue block for microtomyThis course will introduce and review some of the essential background information needed for correct embedding technique. Also discussed in this course will be guidelines for orientation of common histology specimens. Mastery of this information facilitates practice and application of these concepts during execution, to increase the histologist's technical proficiency at paraffin embedding in the histology laboratory.

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Basic Anatomy and Tissue Identification

The embedding histologist must practice to develop rapid visual recognition of tissue types and be able to determine the correct orientation for each specimen using their knowledge of anatomy and tissue sampling techniques. It is often helpful to consider the reason(s) that each specimen may have been selected for submission to histology. Also, remember that clear goals are outlined for each specimen to arrive at a diagnosis based on the type of specimen and the suspected reason for the surgical procedure performed.Gross dissection of tissue samples is determined by a pathologist or pathologist's assistant (PA). Many laboratories follow established protocols for the gross dissection of particular types and categories of specimens. Specimens are identified using a numbering system that associates a unique number with a specific patient, medical record number, and tissue sample upon receipt. The cassette containing the specimen from that unique patient and procedure is also identified with this number. This unique number is transcribed on the slanted top edge of the tissue cassette by either hand or machine, shown in the lower image. The slides prepared from the block in the histology lab from this cassette are likewise identified with this unique number, as shown in the image on the right.Tissue is selected for microscopic examination to document important details about each specimen. Some examples of specimens that may be selected for submission to histology include:All lesions grossly identified - Tissue between lesions may be included with the lesion to determine if the lesion is separate or interconnected to surrounding normal tissue.Representative sections of all normal tissues or structures that are not included with suspected disease-containing sections.All surgical margins, when appropriate.Frozen-section remnants for permanent sections.

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Spatial Reasoning and Visualization

Histology is literally, "the study of tissue." Tissue sections cannot be produced which are completely accurate and representative "snap shots" of the tissue in its' living state. But instead, histologists seek to preserve the tissue in the most life-like state possible. In reality, what we are able to achieve is a somewhat distorted, yet permanent, representation of the tissue's living condition.Histologists must learn to be able to visualize how specimen handling, including gross dissection and tissue processing, affect the appearance of tissues at both the macroscopic and microscopic levels.A clear understanding of anatomy and the ability to spatially visualize the specimen in three dimensions is extremely helpful. The histologist must use this background knowledge and understanding to visual identify any alterations in the tissue appearance caused during gross dissection and/or tissue processing; Apply this insight for correct specimen orientation during embedding.In visually assessing and identifying tissue samples, note that: Some sections cut through tubes or invaginations are not large enough to show the entire lumen. Many cells and tissues are organized into partitions, which divide the structure into numerous smaller areas which may be transected in any given specimen.Many submitted specimens may not show all the structural details that are depicted in images in histology atlases or anatomy textbooks. You may become quite skillful at visually identifying many specimen types, but you MUST still verify the specimen type using an electronic or paper-based worksheet.

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Forceps and Other Tools

Many histologists use forceps and other tools to help them manipulate and position tissue specimens in the desired arrangement. Histologists often have individual preferences for the size and shape of forceps they use. It is recommended that all types of forceps be wiped well between specimens to prevent carry-over contamination. The use of forceps without "teeth" or small grooves at the tip will also help in preventing carry-over. Tissue tampers or stamper can be used to apply pressure evenly on the specimen to help flatten it so that it will harden in one flat plane as the block solidifies. It is essential to make specimens as flat a possible in relationship to the block face for a single, representative section of the entire specimen to be obtained. Forceps and other tools used to manipulate tissue during orientation must be kept warm so that tissue fragments do not adhere to tools as the paraffin on them will begin to solidify on their the surface if they are too cool. Small warming wells are found in most embedding units for the purpose of keeping tools at the necessary temperatures. These wells should be cleaned as frequently as any tools used during embedding, as they can harbor small tissue fragments that can then be transferred to adjacent specimens.

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Which of the following is NOT a tool or instrument utilized for routine paraffin embedding? View Page
Base Molds

Different types of base molds may be used to provide a "frame" for arrangement of the specimen and to hold the molten paraffin while it hardens. The most common type is the metal base mold, but other types such as plastic disposable molds, may be used. The outer dimensions of base molds are standardized to fit within the sizes of most manufacturers' microtome block holders. The block face (indicated by the arrows) is the centrally located, more deeply recessed surface where the tissue specimen will be placed and arranged. This recessed area, which becomes the block face, can be different sizes (shown in the image) to accept differently sized specimens. The base mold size selected should be based on the specimen type and size with consideration of the microtomy protocol that is expected to be performed for that specimen type.

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Cool forceps are recommended for use during paraffin embedding.View Page
Paraffin Temperatures

Paraffin temperatures should be maintained within 2° C to 4° C of the melting point of the paraffin. The measure and control of the temperature that the paraffin media is exposed to during its use should be recorded in the quality control records. The consistent maintenance of temperatures in a controlled range will provide the best results and performance from any paraffin formula selected; since at both on the low and high end of the recommended temperature range, problems can occur. Sectioning of blocks will be easier if the paraffin is cooled at a consistent rate for even solidification of the molten paraffin into its final crystalline structure. Controlled cooling allows the formation of a uniform and homogenous matrix of crystals. Paraffin which cools too rapidly or unevenly can cause grainy textures which interfere with sectioning and may result in artifacts in the final tissue sections. On the other hand, overheating of the paraffin can result in deterioration of the components of the paraffin mixture, and some tissue specimens may become brittle or hardened by prolonged exposure to too hot paraffin.Some technical problems, which are assumed to be microtomy issues, are really problems with media selection or embedding technique, so it is worth the time to investigate.

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Paraffin with a melting point of 55° C has been selected for embedding. When regulating the holding reservoirs in the embedding center, at what approximate temperature should the molten paraffin be regulated? View Page
Basic Tissue Orientation

Many different tissue specimens may be submitted to the typical histology laboratory. Identification of each tissue type is very important for proper embedding orientation. An understanding of the surgical techniques and grossing methods for common specimens will allow visual recognition of the more common tissue types.

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Uncomplicated Specimens

For larger tissue specimens without complicated margins or stratified layers, the orientation is simply to find the cut surface. Most of these types of specimens will be embedded flat on this cut surface so that the cut surface is placed flat against the block face (bottom, most recessed surface of the mold). Your main concerns will be with getting the specimen as flat as possible and leaving a border of paraffin surrounding the outside edge of the tissue. It is helpful to place at a slight angle, any tissue which is dense or rigid in the block face, as this will aid microtomy. Examples of tissue types which might be easiest to cut at a slight angle are uterus body, thyroid, and prostate gland wedges. Large fatty pieces, such as large sections of breast tissue or lipomas, may not have a distinct cut edge, but should be embedded to show the greatest surface area. It is important to also be sure to leave a heavy border of paraffin to help contain the fatty tissue and hold the section together on the water bath during microtomy.

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Small to Medium Sized Specimens with Multiple Pieces

Specimens such as curettage, prostate TURP chips, and other irregularly shaped, multiple piece specimens without clear orientation should be arranged in the block face to show as much surface area of the pieces as possible in the final sections. Try to position each of the pieces to point in the same general direction to reduce as much drag as possible during sectioning, since this can reduce section puckering and wrinkling. You must strive to get all the pieces to harden in the same plane so that one complete, representative section can be obtained.

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Regularly Shaped, Medium Sized Pieces

When embedding medium sized specimens with greater than one piece, locate any inked or cut surface and place all pieces facing in the same direction using these markers.If multiple levels will be cut from the block, select the smallest mold size that will contain the pieces and still allow a border of paraffin on the outside of the specimen. This will make it easier to place more than one section on a slide during microtomy. As with other types of specimens, you will still seek to make sure all the specimens harden in the same plane so that a single representative section is easier to obtain.

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Specimens Containing a Lumen

Any specimen that is a transverse section of a hollow structure or a specimen containing a lumen will almost always be placed in the block face to show the lumen or other opening in cross-section on the final slide. For any organs with very large lumen's in which the tube has been transected, you will embed on the cut surface to demonstrate the lining and layers present. Of special concern are specimens with very tiny lumen openings, such as temporal arteries or vas deferens. Since the lumen opening is so tiny, it is sometimes difficult to see. Some laboratories may lightly dip the end of the tiny tube opening in black ink. This indicates the "end" that should be placed down in the block face. Some laboratories may submit such tiny tubes whole and ask that they be cut into cross-section at embedding. This allows the histologist to clearly see the opening during dissection. Other means to flag these specimens can be by using an abbreviation on the cassette such as "vas" for vas deferens, alerting the histologist that the specimen contained will need to be embedded on end to show the lumen in cross-section.

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Appendix

Appendix specimens submitted to histology will typically include transverse sections of the base and longitudinal sections of the tip (which may be bisected). Orientation will include orientation of any lumen openings of tubular pieces with "on end " orientation to show the lumen openings in cross-section. Any bisected longitudinal pieces should be oriented to show the inner surface and layers present.

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Placenta

Placental specimens submitted to histology may include:Membrane roll: Place on edge perpendicularly to the block face to show membrane layers.Placental disc sections: Embed these flat and centrally located in the block face.Umbilical cord segments: Embed these as other tubes, on end to show the lumen (vessel) openings in cross-section.

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Gynecological (GYN) Specimens

Several types of GYN specimens are routinely submitted to clinical histology laboratories such as cervical loop electro-cautery excision (LEEP) procedures, cervical cone, endometrial curettage, and vulvar biopsies. Although for most GYN specimens you will follow the same general guidelines as for other similarly shaped and sized tissue specimens, it is especially helpful to understand a little about the surgical procedures used to obtain these specimens and also the common methods for dissection used.Cone biopsiesThis procedure is a conical excision of the cervical canal using a laser or cold blade.The wider part of the cone is the ectocervix, and the tapered tip contains the endocervical margin.The ectocervix by convention is described as a clock face, with the most superior midpoint of the anterior lip designated at 12 o'clock.Sections will usually be submitted sequentially and designated in the gross description by their clock face orientation.These sections should be embedded so that perpendicular sections will be shown of the cut surface.Cervical LEEPThese related procedures remove less tissue than a cone biopsy and are obtained by electro-cautery of the cervical transformation zone.The specimen may be divided either perpendicularly or by using a radial dissection method.Endometrial curettageCurettage is a scraping method, in this case of the endometrial lining.The specimen obtained will consists of bloody fragments.These may be submitted in biopsy bags to contain the fragments during tissue processing.The surface of the bags or paper should be scraped lightly to remove as much material as possible.Embed to keep the fragments centralized in the block face and to arrange for the greatest surface area to be shown in one plane.Wipe forceps and all surfaces well after these specimens; it is easy to transfer the loose bloody fragments to other specimens.Embed all fragments with respect to the ink present with inked edges facing all in the same direction.

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Endoscopic Biopsies

Endoscopic procedures yield small pinched off tissue samples and are commonly used for biopsies of the gastrointestinal tract. The specimens obtained from endoscopy will most likely be of small dimensions with difficult to see cut edges.It is unlikely that any inked surfaces will be present. In fact, the entire specimen is often colored with eosin to make it more visible. Since the surface will likely appear homogeneous, you will embed these types of biopsies most often on end, so that the tallest side is traveling into the block interior. This allows the greatest number of sections to be obtained from the tiny specimens for multiple levels, special stains, and immunohistochemistry (IHC). Since multiple level section protocols are very common with these types of biopsies, select the smallest mold size that appropriately contains the specimen pieces.

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Skin Specimens

Specimens of skin will be submitted to histology after being obtained by various surgical biopsy procedures, such as the shave, punch, or curettage methods. In general, skin specimens will be oriented so that sections will be taken at an angle perpendicular to the epidermis. Identification of the epidermis is a good first step in deciding correct orientation of most skin specimens. However, since important diagnostic information can very easily be lost with incorrect orientation of skin samples, it is important to verify the specimen type and biopsy type using the accession log, embedding worksheet, or gross description before proceeding. Do NOT merely rely on visual recognition by itself.

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Elliptical Skin Specimens

Many skin excisions are submitted in an elliptical shape. This is because it creates wounds that lie parallel to the skin tension lines, so the wounds tend to heal with less scaring. Frequently, elliptical skin specimens will have sutures to indicate how the specimen was oriented on the body. Embed these skin shapes so that the cut surface of the tip (broadest surface of the triangle) is down in the block face. This results in successive sections that progress towards the tip and become smaller over multiple sections. This orientation allows the residual tumor, if present, to be traced out to the tiniest point of the tip. The body, or mid sections, of the specimen will be placed on the cut surface to be serially sectioned at an angle perpendicular to the epidermis.

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Needle Biopsies

Needle biopsies are commonly obtained from the prostate or liver. They will resemble long, thin cores of tissue. Due to the very thin diameter typical of these specimens, it is absolutely essential that they be oriented in the same plane, as flat as possible. It is common for multiple level section protocols to be requested as well as special stains with these specimens, so select the smallest mold that is appropriate for the specimen to make it easier to place multiple sections on the same slide. It is best to arrange the long strings in a parallel arrangement, and not randomly with the long strings facing in many directions. Diagonal arrangement can assist if folds or compression are a problem, since this will reduce the distance that the knife blade travels across the specimen.

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Review: General Orientation Guidelines

Specimens with a longer side versus width, such as core biopsies, are ideally arranged in parallel rows perpendicular to what will be the long axis of the slide.Larger specimens should be embedded face up or face down, making sure they lie flat and are in one plane.Multiple fragments of any specimen should be embedded within the same level and in a manner to show the most surface area.Lumen openings must be embedded in cross-section.Stratified layers should be embedded on edge to show all layers.Place at an angle any dense, rigid, or brittle specimens to aid microtomy.Leave a large perimeter of paraffin, especially around fatty specimens.Special orientation instructions are best given in relationship to the block face.If there are questions or concerns about orientation, it is always best to hold the specimen and ask for assistance from the grossing pathologists' assistant (PA) or pathologist to avoid losing important diagnostic information due to incorrect orientation.

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On all of the following specimens, the histologist is given the embedding orientation instructions to "embed on edge." For which specimen is this orientation NOT appropriate?View Page
Fallopian tubes, vas deferens, and blood vessels should be embedded on their SIDES.View Page
Without any other clear orientation triggers, tissues with a cut surface will be oriented so that the cut surface is placed down in the mold.View Page
Communication

Some suggestions for ways to provide orientation instructions for the embedding histologist:Agree as a department (histology staff, supervisors, pathologists' assistants, and pathologists) how you are going to identify and "flag" those specimens needing special orientation.Clearly indicate and seek to standardize, as much as possible, inking patterns and methods of submission for punch, shave, skin ellipse, and tiny lumen specimens.Realize that most histologists will relate the directions given in instructions to the block face. It is helpful to make embedding procedures and protocols as specific as possible, with diagrams that show orientation, arrangement in the block, and other details as the standard operating procedures (SOPs). Define what instructions such as "on edge," "up or down," or "on its side," will mean in your laboratory. This is something that is easy to misinterpret and can mean different things in different laboratories and situations. Train histologists to use the embedding log worksheet and your laboratory information system (LIS), so that they are able to easily find information such as tissue types, number of fragments, and number/letter designations of blocks submitted to histology. Make sure everyone is clear and "on the same page" to save a lot of frustration and possible loss of an irretrievable specimen due to miscommunication and misunderstanding.

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Sub-optimal Specimens

There are a few types of tissue specimens that may be encountered while embedding that will often be less than optimal, despite being processed in an adequate tissue processing program. These specimens include: Fatty tissue: It does not fix well or dehydrate easily and may be "wet" after processing. This will make a white appearance in many specimens when the block is formed due to the water present in the tissue which will not allow the paraffin to infiltrate. Unfixed or poorly dehydrated specimens will often shrink inward from the block face. This tissue may require reprocessing steps with additional fixation, dehydration, and/or wax infiltration.Large tissue: This includes pieces of any tissue type that has been submitted in sizes that are too large for the cassette. The tissue will often not process well, and deep grooves may be pressed into the tissue from the cassette that will have to be sectioned past to obtain a complete section.Specimens with inadequate decalcification: It may be possible to decalcify with decal solutions at your microtome station or use prolonged soaking to help obtain a section.Hair: It simply does not section well. It is best to try to remove hair if possible during gross dissection.Hard foreign materials such as sutures and staples: Items such as this will not section and will tear the tissue section during microtomy. It is best to remove these during gross dissection, but it may be possible to remove during embedding and placed on top of the cassette before it hardens.Sebaceous cyst contents: Quite often, this material does not process or section well. The material can sometimes be removed from inside the cyst and then placed on top of the block so it will not interfere with obtaining a good quality section of the cyst wall. Many of these issues are due to poorly dissected or handled specimens that are then submitted to histology in this less than optimal condition. In other cases, the tissue type itself may present some processing challenges and it may be difficult to optimize processing programs to be optimal for the varied tissue types that most histology laboratories receive. In all instances, you will be attempting to make adjustments and corrections to the best of your abilities for these shortcomings, so that the best possible microscopic section is able to be obtained.

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Basic Quality Control

Histologists must practice to be able to rapidly identify and orient specimens during paraffin embedding. Taking too long to manipulate and position the specimen may cause more than one layer of solidified paraffin to form when molten paraffin is added to paraffin that has solidified in the bottom of the base mold. These separate layers may pull apart during microtomy or storage. Pieces of all types should be placed with intention, in one plane, and not just randomly in the block face. Careless positioning will make it very unlikely that a single representative section can be easily obtained.Correct by re-embedding, any gross defects such as cracks or air bubbles. Observe the specimen for processing issues, such as poor fixation, dehydration, or infiltration. A "white-ish" appearing or "mushy" texture due to inadequate dehydration in a processed tissue block is most likely in need of reprocessing. Submit this block for reprocessing prior to embedding; it is much easier to take the specimen back through these steps for correction now than to work backward from the final cut block.Document and correct labeling discrepancies, illegible blocks, or incompleteness of cases during embedding, rather than "passing" these problems on to the microtomy step.

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A common source of cross-contamination or carry over of specimen fragments to other specimens is the failure to wipe forceps well between separate specimens.View Page
A final microscopic slide shows a tissue section with an incomplete outline that is not representative of the submitted specimen. What is one of the MOST likely causes of incomplete sections that occurs during embedding?View Page

Body Fluid Differential Tutorial
Select the specific cells listed below that can be found in all types of body fluid.View Page

Bone Marrow Aspiration Part I: Normal Hematopoiesis and Basic Interpretive Procedures
Bone marrow Smear Preparation

Accurate interpretation of bone marrow aspirate specimens is highly dependent on two factors: The quality of the sample obtained by the clinician The quality of the prepared bone marrow smears While the technologists have no control over the quality of the aspirate obtained by the clinician, in most cases they are able to impact the quality of laboratory prepared smears.The method used for preparing slides may be dependent on sample volume, staining techniques, storage space, clinical setting, patient status, and hematopathologist's preference. Bone marrow sample preparations can be made on slides or coverslips from a direct marrow sample or a sample that is manipulated to enhance cellularity (selected for marrow fragments or spicules). In situations where the technologist goes to the bedside, touch-preparation (touch-prep) smears from the biopsy core can be prepared before it is placed in fixative.Regardless of which method is the preferred primary technique at your institution, it is useful to be familiar with other methods, since there may be a situation when the use of an alternate method is the only way to obtain an interpretable smear.When adequate samples are provided, it is desirable to make several slides or coverslips so that there will be enough preps available for all the possible tests/special stains that may be requested. The extra smears can be stored at room temperature and protected from light in an envelope or sleeve for further testing, if required.

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Manual Staining of Bone Marrow Preparations: Wright's and Wright-Giemsa Stain

Wright's or Wright-Giemsa stains are usually the preferred staining method for bone marrow aspirate smears. These are methanol-based staining solutions with similar dye composition to the diff-quick stain but require longer stain contact time for adequate staining. The Wright's and Wright-Giemsa stains have a buffer step as well. Since Wright's stains are methanol based they do not require a fixation step prior to staining, although you might prefer to do so first to reduce water artifact that can occur on humid days or with aged stain.In the dip method of staining, the smears are first dipped in methanol to fix the specimens and then placed in Wright's or Wright-Geimsa stain for 10-15 minutes to stain. The smears are next moved to a mixture of stain and 6.8 pH phosphate buffer (usually one part stain to 2-3 parts buffer) and allowed to stain for at 20-30 minutes. After staining, they are given a quick rinse in distilled water and allowed to air dry before mounting or cover-slipping.When using a staining rack, the marrow slides or coverslips are first flooded with enough stain to cover the slide and stained for 10-15 minutes. Then, a 6.8 pH buffer is carefully added without overflowing and gently mixed by blowing until a green metallic sheen forms. This is allowed to stand for 20-30 minutes and then rinsed off with distilled water. The slides or coverslips are then air dried and mounted.Staining times can be extended for extremely cellular marrows; however, care must be taken when using the rack staining method. Extended times can lead to evaporation of the stain and cause excessive precipitation. Both the stain and buffer can be topped up if necessary to prevent this from occuring, while additional rinse time may be needed.Wright's and Wright-Giemsa stains, when performed properly, give sharp and clear nuclear, cytoplasmic, and granule detail. There can be variation in the quality of the stain from batch to batch, dependent on the manufacturer's quality control, storage, and shipping conditions. Many manufacturers age their stains for a minimal amount of time before shipping and assume that there will be additional standing time at the distributor before it reaches your lab. This may work for peripheral blood staining, but it is not ideal for bone marrow staining. It is advisable, if possible, to keep a separate stock of Wright's stain for bone marrow staining which is kept at least 6 months before use. Like a fine wine, the older Wright's stain gets, the better the quality and clarity of the final stain.

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Bone Marrow Delivery

In some institutions, the laboratory technologist does not assist the clinician at the bedside with the bone marrow aspiration procedure. Instead the clinician delivers the bone marrow sample to the laboratory, similarly to other laboratory specimens. When this is the case, the bone marrow sample may be delivered in one of two manners with the laboratory's responsibilities dependent on which method is used. A clinician may deliver to the laboratory a specified number of smears, made at bedside, along with the bone marrow sample. Samples may also be designated for flow cytometry, cytogenetics, or molecular diagnostics. A clinician may deliver a standard package of bone marrow aspirate to the laboratory in various evacuted blood collection tubes. In this situation the laboratory will usually have a standard order set that directs the distribution of the marrow samples based on diagnosis. The hematology laboratory will use these samples to prepare the bone marrow smears, while the other tubes would be distributed for flow cytometry, cytogenetics, molecular diagnostics, etc. based on the direction of the hematopathologist.

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Chemical Screening of Urine by Reagent Strip
Urine Specimen Collection

It is important for a quality urine sample to be sent to the laboratory in order for an accurate set of results to be produced. There are various types of urine samples that may be acceptable for urinalysis in the laboratory. The most common urine collection types are:Random Specimen: This is the specimen type which is sent to the laboratory for analysis most commonly. This type of urine sample is easy to obtain and is also readily available. As the name implies, the random specimen can be collected at any time. Patients should be careful not to touch the inside of the cup or cup lid to avoid any contamination.First Morning Specimen: This type of urine specimen is collected when the patient first wakes up in the morning. This is also occasionally called an 8-hour urine specimen.Midstream Clean Catch Specimen: This type of urine specimen concentrates on the reduction of contaminants in the urine sample by requiring special cleansing protocols. The urine midstream is then collected into a clean container.Other, less common types of urine specimens include:Timed Collection SpecimenCatheter Collection SpecimenSuprapubic Aspiration SpecimenPediatric SpecimenThe general procedure for using a reagent strip is outlined in this exercise. Each test on the strip will be discussed in detail in the remaining exercises.

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All the following statements about the urine specimen are true EXCEPT:View Page
Urine Specimen Processing and Transportation

In order to ensure proper stability of the specimen and accurate test results, there are guidelines in place to aid in the appropriate urine processing and transportation. These guidelines include: Ensuring that all urine collection and/or transport containers should be clean and free of debris or interfering substances.Ensuring that the collection and/or transport container has a secure lid and is leak resistant. Leak-resistant containers reduce specimen loss and healthcare worker exposure to the specimen while also protecting the specimen from contaminants.Utilizing urine containers that are made of break-resistant plastic instead of glass.Utilizing urine containers that do not leach interfering substances into the specimen.Utilizing collection containers and/or transport tubes which will not leak within the pneumatic tube system (if one is used within the laboratory facility). A leak-proof device in this situation is paramount.Proper labeling and correct identification should be applied to the collection container or tubes. This includes noting the time the specimen was collected. Remember that urinalysis specimens must be analyzed within 2 hours of collection.Ensuring that there is sufficient volume to fill the tubes and/or perform the test.

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Clinical Significance of Urine Protein (continued)

Individuals with diabetes mellitus may excrete small amounts of albumin in the urine (microalbumin) which may signal the beginning of reduced glomerular filtration. Stabilizing the blood glucose level at this time may delay progression of diabetic nephropathy. Both type I and type II diabetes mellitus are leading causes of renal failure. Microvascular damage caused by excessive renal exposure to glucose can lead to diabetic nephropathy. By the time the urine protein level reaches the 30 mg/dL level that is necessary for detection by routine reagent strips, damage to the kidneys may have already occurred. Reagent strips are available that use a dye-binding technique rather than the traditional protein-error of indicators principle. These strips are more sensitive and specific for albumin, detecting levels as low as 8 mg/dL.Women in the last month of pregnancy may develop proteinuria as the first sign of impending eclampsia. Eclampsia is the gravest form of toxemia of pregnancy. The presence of protein in this situation must be evaluated by the physician in conjunction with other clinical symptoms.Benign transient proteinuria may be the result of: exposure to cold, strenuous exercise, dehydration, and/or high fever. Benign transient proteinuria may also occur during the acute phase of a severe illness. Patients over the age of 60 have a greater chance of having protein in their urine. Occult malignancies and glomerulonephritis, that occur more frequently in the elderly, may be signaled by the presence of proteinuria. Orthostatic proteinuria is a condition seen most often in young adults. The condition may be caused by pressure on the renal nerve. When this condition is suspected, two urine specimens are tested. One specimen is collected upon arising in the morning, and the second is collected several hours later. When this condition is present, the first morning specimen, after the patient has been in a supine position, will be negative for protein. The second specimen, taken after the patient has been upright for several hours, would be positive for protein.

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Test for Reducing Substances Other than Glucose

Urine specimens from certain pediatric patients should be tested for other reducing substances, such as galactose, when the results for glucose are negative using the routine chemical reagent strip method. The laboratory's procedure should define when additional testing is needed. For example, some laboratories will peform this additional step for each urine specimen received from patients two years old or younger.

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A copper reduction method (e.g. Clinitest® or Benedict's) is performed on pediatric specimens in order to check for the presence of:View Page
False Positive and Negative Ketone Results

False Positive KetonesFalse positive ketone results may be seen in patients after BSP or PSP dye injection due to the phthaleins. The presence of L-DOPA metabolites, some urine preservatives (e.g. 8-hydroxyquinaline), or high levels of phenylketones will also cause false positive results. Antihypertensive drugs such as methyldopa and captopril also may produce false positive results.False Negative KetonesThe presence of excess moisture/humidity can cause the ketone reagent to become nonreactive, resulting in a false negative test for ketones. Urine specimens should not remain at room temperature if testing is delayed because ketones are unstable at room temperature.

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False Positive and Negative Results

False Positives:A false positive result for blood on the urine chemical reagent strip can occur when oxidizing contaminants, such as hypochlorite (bleach), remain in collection bottles after cleaning. Contamination of the urine with provodine-iodine, a strong oxidizing agent, used in surgical procedures can also result in a false positive reaction. Microbial peroxide found in association with urinary tract infections may also cause false-positive results. Capoten® (Captopril) can cause decreased reactivity.The muscle tissue form of hemoglobin, myoglobin is a well-known cause of false-positive reactions on the blood portion of the reagent strip. When tissue hemoglobin is present, the urine specimen has a clear red appearance. Patients suffering from muscle-wasting disorders or muscular destruction due to trauma, prolonged coma, or convulsions or individuals engaging in extensive exertion may have myoglobin in their urine. Specific tests for myoglobin, such as immunodiffusion techniques or protein electrophoresis, are needed to confirm the presence of this substance in a urine specimen. Levels of ascorbic acid normally found in urine do not interfere with this test. False Negatives:False negative results may occur in some analysis methods when the concentration of ascorbic acid is greater than 5 mg/dL. The sensitivity of the blood portion of the test strip is decreased in specimens with a high specific gravity and increased protein. High levels of nitrites may delay the reaction, causing a false negative to be reported. If the pH of a urine sample is below 5, hemolysis of red cells as part of the test reaction is inhibited which results in a false negative reaction. An improperly mixed specimen may test negative if the red blood cells are in the sediment.

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False Positive and Negative Results

False Positives:A false positive nitrite test result may occur when a urine specimen has remained at room temperature for an extended period of time, allowing bacterial contaminants to multiply and produce measurable levels of nitrites. Interference from some medications that cause the urine to become red or orange may lead to an incorrect reading of positive for nitrite. False Negatives:False negative results may occur in urine specimens that did not remain in the bladder for the sufficient length of time needed for the bacteria to reduce a measurable quantity of nitrate to nitrite. Other reasons for false negative results include high specific gravity, ascorbic acid levels above 25mg/dL or a low pH (<6). Less frequently, the cause may be due to a lack of sufficient nitrate in the diet (green vegetables) or further reduction of nitrite to nitrogen when large numbers of bacteria are present. In patients receiving antibiotics, the metabolism of the bacteria may be inhibited which would also produce a false negative reaction.

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Leukocyte Esterase on the Urine Chemical Reagent Strip

If leukocyte esterase is detected on a urine chemical reagent strip, a color change occurs on the reagent pad after the strip is dipped in the urine sample. Be sure to follow the manufacturer's directions for read-time and test interpretation. A positive leukocyte esterase test indicates the presence of granulocytic white blood cells. Lymphocytes do not contain granules, and would not produce a positive leukocyte esterase test. Positive results should be confirmed by performing a microscopic examination on the sediment; keeping in mind that white blood cells may be absent if they are lysed, yet have released their esterases into the urine specimen. Positive results may occasionally be found in random specimens from females due to contamination of the specimen by vaginal discharge.

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False Positive and Negative Urine Leukocyte Esterase

False Positives:A false positive result may occur in the presence of strong oxidizing agents in the collection container. In random urine specimens from women, a positive result for leukocyte esterase may be due to a source external to the urinary tract. Other urine sediment findings such as bacteria, squamous or renal epithelial cells, lymphocytes or red blood cells do not contain esterases, and would not produce a positive leukocyte esterase test. False Negatives:False negative results may occur in the presence of significant levels of protein or glucose and in urines with high specific gravity which can crenate the white blood cells, leaving them unable to release esterases. Some drugs such as Cephalexin (Kelfex®), Cephalothin Keflin®), Tetracycline, or high concentrations of oxalic acid may also cause falsely decrease leukocyte esterase test results.

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Diabetes and the Current American Diabetes Association Guidelines
HbA1C versus Blood Glucose Measurement

Advantages of utilization of HbA1C over blood glucose measurement include: Fasting is not required Greater specimen stability Less fluctuations in day-to-day levels caused by stress and illness Disadvantages of utilization of HbA1C over blood glucose measurement include: Cost per test is higher than blood glucose. Conditions that shorten red blood cell (RBC) survival e.g., hemolytic anemia, homozygous sickle cell trait, pregnancy, or recent significant blood loss, will reduce exposure of RBCs to glucose, thereby lowering the HbA1C test value. Specimens with >10% fetal hemoglobin (HbF) may have a falsely decreased HbA1C test result. If onset of diabetes is rapid, blood glucose levels will more correctly reflect glycemia than HbA1C levels.

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Blood Glucose

Serum, plasma, and whole blood glucose levels are among the most common laboratory assays. Due to self-monitoring of blood glucose (SMBG), blood glucose is also the most common assay performed by patients themselves or their caretakers. Fasting, timed, and casual serum or plasma specimens are run in hospital laboratories for screening, diagnosis, and monitoring of patients.

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Microalbuminuria

Microalbumin is not a measurement of a small size albumin molecule but measurement of low concentrations of urinary albumin in diabetes to identify early renal impairment. Microalbuminuria tests measure concentrations of albumin that are lower than levels that can be detected with routine urine dipstick tests for protein. Timed, overnight, and first morning specimens can be screened for microalbuminuria. Quantitative measurements are also utilized for screening of renal impairment and for monitoring treatment.

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Histology Special Stains: Carbohydrates
Monosaccharides - The Basic Carbohydrate Structure

The simplest form of a carbohydrate is a Monosaccharide, which is the building block of larger and more complex carbohydrates, namely polysaccharides and and glycoconjugates like mucopolysaccharides. Monosaccharidesare not easily demonstrated using histology techniques due to the high number of hydroxyl (OH) groups which render the molecule extremely water soluble. Most monosaccharides that are present within tissue specimens will be lost during the fixation and standard tissue processing. Glucose, is the most commonly demonstrated monosaccharide in the histopathology laboratory.

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Human Papillomavirus (HPV) and Molecular Diagnostic Testing
Molecular Detection of HPV in Cervical Cytology Specimens

HPV molecular testing involves the detection of HPV viral DNA in cervical cells collected during a pelvic examination. Molecular testing is utilized for detecting high risk-HPV (HR- HPV) types. The testing can be performed as: Primary screening for HPV with or without a Pap test Follow-up on abnormal Pap smear cytology Triage of atypical squamous cells of undetermined significance (ASC-US) Pap smear results

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Molecular Methods for HR-HPV Testing

The molecular methods currently used for HR-HPV testing include: Polymerase chain reaction (PCR) PCR is the molecular biology technique used to type HPV. There are also PCR tests to detect HR-HPV DNA. In situ hybridization (ISH) and Fluorescence in situ hybridization (FISH) These methods use chromagens or fluorescence labeled probes to detect HR-HPV DNA in tissues or cells on planar surfaces. Digene High-Risk HPV Hybrid Capture (HC) 2 DNA Cervista HPV HR Screen and Cervista HPV 16/18This study will discuss the last two methods. High-Risk HPV HC2 DNA and the Cervista HPV assays are FDA-approved for laboratory testing and are designed as molecular assays for the clinical laboratory. These two methods are most often assayed using the liquid cervical specimens collected during the Pap smear procedure.

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Twenty-five samples of isolated DNA from liquid cervical cytology specimens, one negative control, and one positive control were assayed with the High-Risk HC 2 DNA assay. All patient samples and both controls were positive for HR-HPV types.Which one of the following is likely the cause of the false positives?View Page
References

Cervical Cancer: Prevention and Early Detection. American Cancer Society. Available at http://www.cancer.org/docroot/CRI/content/CRI_2_6x_cervical_cancer_prevention_and_early_detection_8.asp. Accessed December 1, 2011. Cervista HPV, Cervista HPV – Invader Technology. HOLOGIC. Available at http://www.cervistahpv.com/laboratory/invadertechnology.html. Accessed December 1, 2011.Chin-Hong PV, Klausner JD. Diagnostic tests for HPV infection. Medical Laboratory Observer. October 2004:10-16.Cobo F, Concha A, Ortiz M. Human papillomavirus (HPV) type distribution in females with abnormal cervical cytology. A correlation with histological study. Virology Journal. 2009;3:60-66.Cox JT, Moriarty AT, CastlePE. Commentary on statement on HPV DNA test utilization. American Journal Clinical Pathology. 2009;131:770-773.HPV Vaccine Information for Clinicians. Centers for Disease Control and Prevention. Available at http://cdc.gov/std/hpv/stdfact-hpv-vaccine-hcp.htm. Accessed December 1, 2011.Human Papillomavirus (HPV) Natural History. American Society for Colposcopy and Cytological Pathology. Available at http://www.asccp.org/hpv_history.shtml. Accessed December 1, 2011.Human Papillomavirus (HPV) Vaccines. National Cancer Institute. Available at http://www.cancer.gov/cancertopics/factsheet/prevention/HPV-vaccine. Accessed December 1, 2011.Human papillomaviruses and Cancer: Questions and Answers. National Cancer Institute Fact Sheet. Available at http://www.cancer.gov/cancertopics/factsheet/risk/hpv. Accessed December 1, 2011.Hybrid Capture 2 Technology. QIAGEN - Sample & Assay Technologies. Available at http://www1.qiagen.com/hpv/hc2technology.aspx. Accessed December 1, 2011.Markowitz LE, Sternberg M, Dunne EF, et al. Seroprevalence of human papillomavirus types 6, 11, 16, and 18 in the United States: national health and nutrition examination survey 2003-2004. Infectious Disease. 2009;200:1059-1067.Molecular Diagnostics Fundamentals, Methods, and Clinical Applications. Leal Buckingham and Maribeth L. Flaws. Philadelphia:FA Davis Company, 2007.Schutzbank TE, Jarvis C, Kahmann N, et al. Detection of high-risk papillomavirus DNA with commercial invader-technology-based analyte-specific reagents following automated extraction of DNA from cervical brushings in Thinprep media. Journal of Clinical Microbiology. 2007;45:4067-4069.Solomon D, Papillo JL, Davey DD. Statement on HPV DNA test utilization. American Journal of Clinical Pathology. 2009;131:768-769.Vernick JP, Steigman, CK. The HPV DNA virus hybrid capture assay: what is it—and where do we go from here? Medical Laboratory Observer. Mar 2003:8-13.Voss JS, Kipp BR, Campion MB et al. Comparison of fluorescence in situ hybridization, hybrid capture 2 and polymerase chain reaction for the detection of high-risk human papillomavirus in cervical cytology specimens. Analytical and Quantitative Cytology and Histology. 2009;31:208-216.

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Molecular Methods in Clinical Microbiology
Human Papilloma Virus (HPV) and Mycobacterium

Human papilloma virus (HPV) is estimated to be the most common sexually transmitted infection in the United States. Digene's hybrid capture assay for HPV received approval by the Food and Drug Administration (FDA) in 2003. Only in recent years have other manufacturers, such as Third Wave Technologies, added this virus to their testing capabilities.Mycobacterium species represented another desirable target for molecular testing. Although some improvements in cultivation and staining techniques had been realized through the incorporation of broth media and fluorochrome staining, identification is still hampered by the growth rate of the organism. Gen-Probe first marketed probes that would allow identification of tuberculosis, M. avium-intracellulare, and M. gordonae in culture positive specimens. These probes greatly streamlined the workup of culture positive specimens.Of great interest to both clinicians and infection control practitioners, is the direct detection of Mycobacterium in clinical specimens. Gen-Probe received FDA approval for its AMPLIFIED MTDâ product for this specific application (in smear positive specimens) in 1995. This method employs isothermal transcription mediated amplification; the amplicons are detected using the same hybridization as the culture confirmation tests.

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The Key Benefits: Specificity of Identification

Even if an organism can survive various transport conditions and will be recovered in available culture systems with reliability, identification protocols may not provide clear cut differentiation, based on phenotypic tests and characteristics. Sequencing techniques are increasingly utilized for certain classes of organisms. Good examples are the aerobic, non-spore forming gram positive rods, including Nocardia and related genera, and mycology isolates. A more routinely encountered application occurs in virology. Molecular methods allow for efficient typing of herpes simplex (type I versus type II) and subtyping of specimens positive for influenza A.

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The Key Benefits: Reduced Turnaround Time

Organisms that are either present in very low numbers, or that possess a characteristically slow growth rate, may require an extended incubation before they are detected in culture. Amplification and/or detection of unique sequences of either DNA or RNA provide for a more timely identification. This is true whether they are applied to the specimen for direct detection, or in some cases, to culture positive specimens for culture confirmation/identification.Even for organisms that are easy to recover and identify (example: Staphylococcus aureus) various molecular methods offer the ability for either direct detection in clinical material, or more rapid identification that would greatly aid in treatment and/or clinical management decisions.

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Why can molecular methods offer improved turnaround times over cultivation methods? (Choose all that apply.)View Page
Challenges for Implementation: Separation of Key Activities

Prevention of contamination is a very key consideration when introducing molecular methods into the routine diagnostic laboratory setting. Because amplification methods are so sensitive, the incidental introduction of even a few copies of exogenous nucleic acid can lead to false positive results. Both physical design and process controls are key aspects of preventing erroneous results.Significant potential sources of contamination are the large quantities of target molecules from previously amplified materials. Amplicons may contaminate work surfaces, air space, pipettes, and reagents. Scrupulous technique is one aspect of preventing contamination; another is the physical separation of specific steps of the process. Ideally, all molecular work will take place in distinct areas designated for key aspects of the workflow, with each area having its own dedicated equipment (especially pipetting equipment). The three designated areas are:Clean area: Where master mixes and other reagents are prepared in the absence of any specimen material. In this area, protection from aerosol contamination is a key consideration. Use of a dead air box with ultraviolet (UV) lighting for decontamination, or a separated area with controlled airflow, are two ways to address this need.Specimen preparation and extraction area: Separated from the area where amplification and detection of amplified product occurs, in order to prevent contamination of samples with amplicons of previously processed specimens.Amplification, detection, and identification of the amplified product: Ideally, this area would be both separated and enclosed.To some extent, the introduction of platforms that utilize automated specimen processing equipment and/or closed amplification and detection systems mitigates stringent separation and space requirements. Good practice, however, would always include designated spaces for each activity, as well as a defined workflow.

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Challenges for Implementation: Workflow Requirements

However limited or spacious the available area is, the establishment of a work process that directs the flow of both staff and specimens in a unidirectional flow is essential. Personnel should be educated to begin work in the "clean" area designated for reagent preparation; move on to the area designated for specimen preparation and extraction, and lastly, proceed to the area for amplification and detection.Back and forth movement between these areas in the course of one run of specimens should NOT occur. Each area should have the necessary dedicated equipment to allow for the segregation of activities to their designated areas. Examples include pipette devices, small tabletop centrifuges, water baths, and racks. Gloves should be removed and disposed of before proceeding from one area to the next.

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Challenges for Implementation: Required Work Skills

In addition to instilling a consciousness about movement from one area to the next, other work skills are critical and key to obtaining accurate results:Aseptic techniquePipetting skillsAseptic technique Due to the sensitivity of molecular assays, there is little margin for error. Cross contamination from specimens with large numbers of organism/target nucleic acid can result from the slightest deviation in aseptic technique. The use of aerosol barrier pipette tips and positive displacement pipette devices go a long way in preventing aerosol contamination, but do not replace the attentiveness of the technologist during each transfer of specimen material. When working with specimens, only one specimen container should be opened at a time.Pipetting skills Although more and more assays are being introduced with pre-prepared master mixes, invariably some reagent preparation is required. This often entails the transfer of very small volumes, which leaves no margin of error during pipetting. Attentiveness to accuracy of pipetting is a must.

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Why is it important to consider work space and workflow design for molecular methods? (Select all that apply.):View Page
Molecular Versus Culture - Pros and Cons

Traditional culture methods for the detection and identification of methicillin-resistant Staphylococcus aureus (MRSA) employing mannitol salt and/or blood agar for cultivation, can take up to 72 hours for isolation and identification, depending on the identification procedures utilized. Concurrent with the development of molecular assays, improvements in culture methods have also been achieved. CHROmagar™ media, specific for MRSA, are employed by many laboratories. These media are both selective and differential, containing chromogenic substrates. MRSA strains utilize the substrates to produce colonies of a specific and characteristic color, minimizing the need for additional identification procedures.Initially these agars required 48 hours of incubation; newer formulations require only 24 hours incubation.Given the reduced incubation and identification requirements, what are the pros and cons of the molecular assays? Cost per test will be greater with the molecular assays as compared to culture methods. Will molecular methods provide for a more efficient workflow and significant improvement in availability of results? To some extent, this will depend on how they can be implemented within each different laboratory setting. Both of the previously described molecular assays require manual specimen preparation and extraction before the sample is placed into the instrument. This hands-on work may actually be greater than the effort expended in swabbing and streaking a culture plate. How much an obstacle this is for implementation will depend on both the volume of testing and the staff available. In a high volume setting, this will be a greater factor.Will tests be performed as specimens come in, or will specimens be accumulated and batched? If controls are required with each run, batching is desirable to reduce this cost. If testing will occur in batches, how many batches can be performed in one day? This will be heavily influenced by the capacity of the instrument. (For example, a single Smart Cycler unit can run up to 16 samples; multiple units would be needed in a high volume lab.) Can they be set up on more than one shift? The greater the number and frequency of batches that can be run, the greater improvement in turnaround time can be realized. Given these variables, implementation of a molecular assay for MRSA is not a given in each laboratory.

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Current and Future Prospects

Cepheid's next generation platform, the Xpert® system, addressed some of the problematic factors by simplifying the workflow and making on-demand testing feasible. The assay targets the SCCmec-orfX junction, and utilizes a self-contained cartridge containing all the reagents necessary for real time PCR. Swabs are placed into an elution fluid and vortexed; an aliquot is dispensed into a reagent cartridge, which is installed on the Xpert platform. Results are available in approximately 75 minutes. Capital investment for equipment and ongoing reagent costs remain a consideration with this system. Another consideration for laboratories is throughput. Although the Xpert® platform offers excellent turnaround time, the largest system has 16 modules. Facilities that need to process very large volumes of specimens will require multiple modules, adding to capital considerations.Large volume platforms that automate sample preparation, extraction, amplification, and detection in a real time format would represent the ideal, walk-away solution for many laboratories, as well as a target for development for manufacturers.

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References

BD GeneOhm™ MRSA [package insert]. Quebec, Qc, Canada: BD Diagnostics; 2009. Available at: http://www.bd.com/geneohm/english/products/pdfs/mrsa_pkginsert.pdf. Accessed February 22, 2012.Bonetta L. Prime time for real-time PCR. Nature Methods. 2005;2:305-312. Available at: http://www.nature.com/nmeth/journal/v2/n4/full/nmeth0405-305.html. Accessed February 22, 2012.Boughton B. Universal PCR Screening for MRSA May Cut Costs, Reduce Infection. In Medscape Medical News. Available at: http://www.medscape.com/viewarticle/708813. Accessed February 22, 2012.CDC Response: A Year in Review. Centers for Disease Control and Prevention Website. Available at: http://www.cdc.gov/h1n1flu/yearinreview.htm. Accessed February 22, 2012.Centers for Disease Control and Prevention. Evaluation of Rapid Influenza Diagnostic Tests for Detection of Novel Influenza A (H1N1) Virus ---United States, 2009. Morbidity and Mortality Weekly Report. August 7, 2009;58(30):826-829. Available at: http://www.cdc.gov/mmwr/preview/mmwrhtml/mm5830a2.htm. Accessed February 22, 2012.Department of Biochemistry. University at Buffalo, School of Medicine and Biomedical Sciences Website. Available at: http://www.smbs.buffalo.edu/bch/Labs/SinhaLab/Protocols/RT-PCR.pdf. Accessed February 22, 2012.Desjardins M, Guibord C, Lalonde B, Toye B, Ramotar K. Evaluation of the IDI-MRSA Assay for the Detection of Methicillin-Resistant Staphylococcus aureus from Nasal and Rectal Specimens Pooled in Selective Broth. J Clin Microbiol. 2006 April;44(4):1219-1223. Available at: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1448652/. Accessed February 22, 2012.Eastwood K, Else P, Charlett A, Wilcox M. Comparison C. difficile detection methods. J Clin Microbiol. 2009;doi:10.1128/JCM.01082-09. Available at: http://jcm.asm.org/cgi/content/short/JCM.01082-09v1Farley JE, Stamper PD, Ross T, Cai M, Speser S, Carroll KC. Comparison of the BD GeneOhm Methicillin-Resistant Staphylococcu aureus (MRSA) PCR Assay to Culture by Use of BBL CHROMagar MRSA for Detection of MRSA in Nasal Surveillance Cultures from an At-Risk Community Population. J Clin Microbiol. 2008;46(2):743-746. Available at: http://jcm.asm.org/content/46/2/743.full. Accessed February 22, 2012.Forrest GN, Mehta S, Weeks E, Lincalis DP, Johnson JK, Venezia RA. Impact of Rapid In Situ Hybridization Testing on Coagulase Negative Staphylocci Positive Blood Cultures. J Antimicrob Chemother. 2006;58(1):154-158. Available at: http://jac.oxfordjournals.org/content/58/1/154.full. Accessed February 22, 2012.Garcia LS, Isenberg HD, eds-in-chief. Clinical Microbiology Procedures Handbook. 2nd ed. Washington, DC: ASM Press; 2007.Hindiyeh M, Hillyard DR, Carroll KC. Evaluation of the Prodesse Hexaplex Multiplex PCR Assay for Direct Detection of Seven Respiratory Viruses in Clinical Specimens. Am J Clin Pathol. 2001;116:218-224. Available at: http://ajcp.ascpjournals.org/content/116/2/218.full.pdf. Accessed February 22, 2012.Hunt M. Real Time PCR. University of South Carolina School of Medicine Website. Available at: http://pathmicro.med.sc.edu/pcr/realtime-home.htm. Accessed February 22,2012.Interim Guidance for Influenza Surveillance: Prioritizing RT-PCR Testing in Laboratories. Centers for Disease Control and Prevention Website. Available at: http://www.cdc.gov/h1n1flu/screening.htm. Accessed February 22, 2012.Interim Guidance for the Detection of Novel Influenza A Virus Using Rapid Influenza Diagnostic Tests. Centers for Disease Control and Prevention Website. Available at: http://www.cdc.gov/h1n1flu/guidance/rapid_testing.htm. Accessed February 22, 2012.Levenson D. Molecular Testing for Respiratory Viruses. In Clinical Laboratory News. March 2008: Vol 34, No 3. Washington, DC: AACC Press; 2008. Available at: http://www.aacc.org/publications/cln/2008/mar/Pages/cover1_0308.aspx. Accessed February 22, 2012.Morshed MG, Lee MK, Jorgensen D, Issac-Renton JL. Molecular methods used in clinical laboratory: prospects and pitfalls. FEMS Immunol Med Microbiol. 2007;49:184-191. Available at: http://www.canlyme.com/morshed_pcr.pdf. Accessed February 22, 2012.Paillard F, Hill CS. Direct nucleic acid diagnostics tests for bacterial infectiousdiseases: Streptococcal pharyngitis, pulmonary tuberculosis, vaginitis, chlamydial and gonococcal infections. MLO-online. 2004;10-15. Available at: http://www.mlo-online.com/articles/0104/mlo0104coverstory.pdf. Accessed February 22, 2012.PCR: an outstanding method. Roche Website. Available at: http://www.roche.com/pages/facets/pcr_e.pdf. Accessed February 22, 2012.Persing DH, ed-in-chief.Molecular Microbiology, Diagnostic Principles and Practice. 2nd ed. Washington, DC: ASM Press; 2010.Pfaller MA. Molecular Approaches to Diagnosing and Managing Infectious Diseases: Practicality and Costs. Emerg Infect Dis. 2001;eid0702. Available at: http://wwwnc.cdc.gov/eid/article/7/2/70-0312_article.htm. Accessed February 22, 2012.Rossney AS, Herra CM, Brennan GI, Morgan PM, O'Connell B. Evaluation of the Xpert Methicillin-Resistant Staphylococcus aureus (MRSA) Assay Using the GeneXpert Real-Time PCR Platform for Rapid Detection of MRSA From Screening Specimens. J Clin Microbiol. 2008;46(10):3285-3290. Available at: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2566096/. Accessed February 22, 2012.The 2009 H1N1 Pandemic: Summary Highlights, April 2009-April 2010. Centers for Disease Control and Prevention Website. Available at: http://www.cdc.gov/h1n1flu/cdcresponse.htm. Accessed February 22, 2012.Timeline of PCR and Roche. Roche Website. Available at: http://molecular.roche.com/About/pcr/Pages/PCRTimeline.aspx. Accessed February 22, 2012.

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Multi-drug Resistant Organisms: MRSA, VRE, and Clostridium difficile
Clostridium Species

Clostridium are gram-positive or gram-variable, spore-forming, catalase-negative anaerobic bacilli. More than 100 species are currently recognized, though relatively few are encountered in properly collected clinical specimens from humans. There are three types of infection associated with Clostridium species: Non-invasive: Toxin-mediated Invasive: Progressive infection with tissue destruction Purulent disease: Closed space (e.g., in the peritoneal cavity) mixed infection with multiple organisms.Clostridium are well known as the agents of these classic toxin-mediated diseases : DISEASE TOXIN INVOLVED CAUSATIVE ORGANISM Tetanus or "lock jaw" Tetanospasmin Clostridium tetani Myonecrosis/Gas gangrene Exotoxins Clostridium perfringens Botulism (severe food poisoning) Botulin Clostridium botulinum

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Screening Cultures for Vancomycin-Resistant Enterococci

Many hospitals have chosen to implement screening programs to identify patients who are colonized with vancomycin-resistant enterococci (VRE). Screening patients provides information about potential source of illness and allows staff to implement appropriate infection control measures. These measures decrease transmission and reduce the number of patients who become infected with VRE.Peri-rectal or anal swabs, as well as stool specimens, are inoculated onto selective media. One medium utilized is bile esculin azide agar containing 6µg/mL of vancomycin. Black colonies that grow on this medium are identified as Enterococcus to the species level and further confirmed as vancomycin-resistant by an appropriate susceptibility testing method.Chromogenic agars specific for VRE are also commercially available.

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Preliminary Identification of the Primary Select Agents of Bioterrorism
Definition of a Sentinel Laboratory

Laboratories within the LRN are divided into 3 levels: Sentinel labs Reference labs National labsAny laboratory that performs microbial analysis is by default a sentinel laboratory. A laboratory that refers specimens that may contain microbial agents or toxins is also a sentinel laboratory. These laboratories are the first responders in the line of defense. Based on the extent of service provided, sentinel laboratories are designated as basic or advanced.

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Role of a Sentinel Laboratory

The role of a sentinel laboratory is to utilize standardized testing protocols to rule-out critical biological agents and refer them to one of the Laboratory Response Network (LRN) reference laboratories, such as public health laboratories, for confirmation. In the event that a potential agent of bioterrorism is suspected, the appropriate internal staff should be notified and then contact your LRN reference laboratory. The sentinel laboratory should NOT: Make the decision that a bioterrorism event has occurred Contact law enforcement or public health officialsSentinel laboratories are divided into basic and advanced categories based on the level of diagnostic testing performed. Advanced sentinel laboratories have greater capability to analyze specimens that may contain an agent of bioterrorism than a basic level laboratory would have.

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Basic Sentinel Laboratory within the Laboratory Response Network (LRN)

A basic sentinel laboratory has the following characteristics: Certified by the Centers for Medicare & Medicaid Services (CMS) for subspecialty within the specialty of microbiology Accredited by Clinical Laboratory Improvement Amendments of 1988 (CLIA) or a deemed status agency Has policies and procedures in place for the referral of diagnostic specimens to an advanced sentinel laboratory Has policies and procedures in place for the direct referral of suspicious isolates to an appropriate LRN reference laboratory

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Any laboratory that analyzes microbes or refers specimens that have the potential to contain microbial agents or toxins is a sentinel laboratory.View Page
Protocols

The American Society for Microbiology (ASM) has developed standardized guidelines in coordination with the Centers for Disease Control (CDC) and the Association of Public Health Laboratories (APHL). These protocols should be integrated into the standard operating procedures of any sentinel laboratory. The purpose is to provide the algorithms used to rule out suspected critical agents of bioterrorism and to refer the specimens to public health laboratories for confirmation. The protocols are available to sentinel laboratories at: http://www.asm.org/index.php/policy/sentinel-level-clinical-microbiology-laboratory-guidelines.html

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Bacillus anthracis

Clinical specimens where organism may be encountered: CSF Blood Stool (rare) Vesicle fluid, skin swab, or biopsy Gram stain morphology from clinical specimens: Large, gram-positive rods with square or concave ends in short chains Spores are usually NOT present Capsule may be viewed in smears from infected tissue, but this is NOT reliable Gram stain morphology from culture material: Large, gram-positive rods with square or concave ends, often in long chains (more than 2-4 cells) Cells easily decolorize as the culture ages Does NOT form capsules in culture Central to sub-terminal, oval spores, with NO significant swelling of the cell It must be noted that spore production increases with the age of the culture. Do NOT keep these cultures in the laboratory for longer than 24 hours for this reason!

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Yersinia pestis

Clinical specimens where organism may be encountered: Blood Lymph node aspirate Respiratory secretionsGram stain morphology: Gram-negative rod Resembles other Enterobacteriaceae Can form short chains Gram stains performed from blood culture or other liquid media may show bipolar staining (displayed by the arrows)Note: Use of Wright-Giemsa staining on direct specimen may enhance demonstration of characteristic bipolar staining, also referred to as "safety-pin" morphology. Use of this staining is of limited value, as the method is not very sensitive or specific.

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Francisella tularensis

Clinical specimens where organism may be encountered: Blood Biopsy, skin scraping, or swab Lymph node aspirate Respiratory secretions - oropharyngeal aspirate, sputum, or bronchial washingsGram stain morphology: Very tiny, gram-negative coccobacillus Pale or weak staining Due to the small size, often difficult to see individual cells

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Brucella species

Clinical specimens where organism may be encountered: Blood Bone marrow TissueGram stain morphology: Very small, gram-negative coccobacilli Stains very faintly and tends to retain crystal violet, especially in blood cultures May initially be identified as gram-positive Organism is larger than F.tularensis Individual cells are evident

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Burkholderia species

Clinical specimens where Burkholderia species may be encountered: Blood Bone marrow Respiratory specimens - sputum, throat, or nasal Wounds UrineGram stain morphology:B. mallei Gram-negative coccobacillus or small rod Arranged in pairs end-to-end, parallel bundles, or Chinese letter formB. pseudomallei Small, straight, or slightly curved gram-negative rod May demonstrate peripheral or bipolar staining as they age (appear like endospores) Smooth forms are arranged in long, parallel bundles Rough forms more irregularly arranged

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Gram stains are performed on positive blood culture bottles. Match the organism that MOST closely resembles the description of the Gram stain morphology provided.View Page
Toxins

Toxin Comment Most Likely Means of Dissemination Primary Route of Entry General Signs and Symptoms Laboratory Testing Botulism toxin: Gram stained image of C. botulinum courtesy of CDC Produced by Clostridium botulinum Could be purified and used in a bioterrorist event to contaminate food or aerosolized to cause disease Aerosol Food contamination Inhalation Ingestion Difficulty speaking or swallowing Blurred or double vision Drooping eyelids (ptosis) Dilated pupils Dry mouth, decreased gag reflex Weakening of the reflexes (hyporeflexia) Abnormal sensations such as numbness, tingling, and progressive arm or leg weakness Flaccid paralysis Culture, anaerobic Digoxigen-labeled IgG ELISA to detect A, B, E, and F toxins Mouse Bioassay for all toxin types and to confirm DIG ELISA Ricin toxin: Extracted from Castor beans Inhibits protein synthesis Causes death approximately 72 hours after initial exposure As an aerosol Inhalation Fever Cough Chest tightness Dyspnea Cyanosis Gastroenteritis Necrosis Antibody detection in clinical specimens Clinical testing not performed unless known exposure has occurred

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Role of a Sentinel Laboratory Regarding Toxins and Viruses

Always follow your facilities procedures and contact your Laboratory Response Network (LRN) reference laboratory for guidance with regard to the toxins and viruses that may be used as bioterrorism agents. A sentinel laboratory should NOT attempt to: Accept or process environmental or animal samples Culture from clinical specimens or detect for these agents Collect specimens for suspect viruses unless directed by a public health officialThe laboratory testing listed on the following pages is intended ONLY as information. For suspect specimen collection, contact your LRN reference laboratory for guidance before collecting or referring specimens.

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A sentinel laboratory should NOT accept orprocess environmental or animal samples or culture clinical specimens for viruses or toxins that are potential agents of bioterrorism.View Page

Reading and Reporting Gram Stained Direct Smears
What is the Value of a Direct Smear?

Gram stain is used primarily as a differential stain for bacteria, although it will also stain most fungi (especially yeasts) and some parasites, including Strongyloides and Trichomonas. The Gram stain procedure is commonly performed on direct smears of clinical specimens and on smears from cultures. This course will focus on Gram-stained direct smears. A direct smear from a clinical specimen can be used to: Judge the quality of the specimen. Provide the clinician with same-day information regarding possible pathogenic organisms, pending results of culture and sensitivity. Contribute to selection of culture media, especially with mixed flora. Provide internal quality control when direct smear results are compared to culture results.

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Cerebrospinal Fluid and Specimens Collected from Other Sterile Sites

Cerebrospinal fluid (CSF) and all specimens collected from sterile sites should have a microscopic examination performed along with culture. Bacteria found in CSF, blood, tissue, and specimens from other sterile sites are always significant.CSF should be cytospun, if possible, to increase the chance of detecting a pathogen. The quantity of organisms seen and the amount and type of host cells, e.g., mononuclear or polymorphonuclear (PMN) white blood cells, is important to report. The presence of PMNs indicates bacterial infection. It is also important to determine and report whether the bacteria are found inside or outside of white blood cells.

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Correlate Direct Smear Results With Culture Results

Correlation between a direct Gram-stained smear of the specimen and what grows out in culture should always be attempted. However, some bacterial organisms may appear differently when viewed in direct clinical specimens versus isolates growing on solid or in liquid culture media. For example, if gram-negative organisms are surrounded by large amounts of cellular material in the direct smear, the cellular material may totally or partially prevent decolorization of the bacteria so that the gram-negative bacteria growing in culture may appear gram-positive or gram-variable on direct smear.

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Size and Appearance of Nonbacterial Cellular Elements on Gram Stained Smears

Type of Cell Average Size Image Comments Epithelial cells 25 µm Appear pink/red on Gram stained smear. Larger than white blood cells. Have a single nucleus. They are an indication of a suboptimal or unacceptable specimen if present in large numbers in sputum specimens, tracheal or endotracheal aspirates, or in urine specimens. White blood cells 12 µm Appear pink/red on Gram stained smear. Most often, polymorphonuclear white blood cells (PMNs). White blood cells indicate inflammation and possible infection. The direct smear examination should focus within and around these cells. Hyphae/pseudohyphae Varies Appear blue on Gram stained smear. Hyphae are tubular filamentous fungal elements, which may show branching or intertwining. Pseudohyphae are multiple buds of yeast that do not detach, thereby forming chains. Yeast 7 µm Appear blue on Gram stained smear. Round to oval, often budding. About the same size as red blood cells. Generally much larger than bacteria. A few yeast may be present as normal flora in upper respiratory tract or genital tract. They may be significant if they predominate, or if budding yeast forms are seen. Red blood cells 7µm Appear red on Gram stained smear Not usually considered a significant finding.

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Significance of Specific Findings

When evaluating Gram stains of clinical samples, keep in mind the source of material from which the smear was made. Bacteria found in cerebrospinal fluid (CSF), blood, tissue and specimens from other sterile sites are always significant. Gram stains of body fluids that are normally sterile must be examined carefully. For every one organism per oil immersion field, there are about 105 organisms per mL present in the sample! Examining stained smears of CSF sediment may assist the clinician in establishing a presumptive diagnosis. The Gram stain result and the results of other special stains could also guide in the selection of culture media. If bacteria are observed in a CSF specimen, it is important to determine and report whether the bacteria are inside or outside of white blood cells (intracellular or extracellular). The quantity of organisms seen and the amount and type of host cells are also important to report. Bacteria observed in specimens from the throat, genital tract and other areas containing normal flora suggest infection only if their composition and type varies significantly from the norm.

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Quality Control Smears

According to regulatory requirements, Gram stain quality control (QC) testing must be performed with each new batch of stain and at least weekly, using both gram-positive and gram-negative organisms. However, individual laboratories may have more stringent requirements. No matter what the required frequency for QC testing, if you prepare and read Gram stains infrequently it would be helpful to stain quality control slides at the same time as a patient Gram stain smear. The image on the right represents control slides using Staphylococcus aureus as the gram-positive control and Escherichia coli as the gram-negative control. If properly stained, the Staphylococcus aureus on the positive control slide (side A in the image) will stain blue, or gram-positive. The Escherichia coli on the negative control slide (side B in the image) will stain red, or gram-negative. If these slides are not stained properly, the problem must be resolved and new slides made before patient specimens are examined and reported.

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Special Topics in Phlebotomy
Become and Remain a Competent Professional

Phlebotomy is a skill that needs to be perfected. Because phlebotomy is an invasive procedure, it is imperative that you become and remain proficient. Many people are apprehensive when getting their blood drawn. Your professionalism will greatly decrease their fears.Individuals who collect blood specimens should be assessed for competence by a qualified mentor before being allowed to perform procedures on patients and periodically thereafter. It is important to receive feedback from the instructor/mentor so that you are ensured your phlebotomy techniques are appropriate. Any remedial training needed should be provided by qualified instructors in a controlled environment--preferably a classroom and not in the presence of patients. Training and competency assessment should again occur whenever new equipment is introduced. Training and assessment records should be kept in your employee file.Ask for assistance when unsure about a collection. Be professional at all times. You are an important part of the health care team.

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References

Clinical and Laboratory Standards Institute (CLSI). Procedures for the Collection of Diagnostic Blood Specimens by Venipuncture; Approved Standard. 6th ed. CLSI document H3-A6. Wayne, PA: CLSI: 2007.Clinical and Laboratory Standards Institute (CLSI). Procedures and Devices for the Collection of Diagnostic Capillary Blood Specimens; Approved Standard. 5th ed. CLSI document H4-A5. Wayne, PA: CLSI: 2004. Clinical and Laboratory Standards Institute (CLSI). Procedures for the Handling and Processing of Blood Specimens; Approved Guideline. Third Edition. CLSI document H18-A3. Wayne, PA: CLSI: 2004.Ernst DJ. Applied Phlebotomy. Baltimore, MD: Lippincott Williams & Wilkins: 2005.

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Introduction

Patient-centered health care is care that is delivered in a manner that is "respectful of and responsive to individual's preferences, needs, and values."*Great health care for every patient involves a team approach. All team members contribute in a unique way to ensure successful patient outcomes. The phlebotomist is a key member of the health care team and the team relies on the phlebotomist to obtain quality specimens. Patient diagnosis and treatment is often dependent on laboratory test results. The accuracy and reliability of these results are contingent on a quality specimen. It is easy to see how the phlebotomist directly affects the care of the patient. As members of a professional health care team, phlebotomists should exhibit professional behaviors. Simple things, such as appropriate dress and grooming, reflect a professional image. Language and conversation should also show that you value yourself, your employer, and the patient. Work habits demonstrate to the rest of the team that you provide an invaluable service.*Reference: Committee on Quality of Health Care in America. Crossing the Quality Chasm, A New Health System for the 21st Century. Washington, DC: National Academy Press. 2001.

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Screening for Diabetes Mellitus and Gestational Diabetes

Glucose tolerance tests are used to help diagnose diabetes mellitus or gestational diabetes, which occurs during pregnancy. The procedure basically consists of these steps:Confirm that the patient has been fasting.Collect a fasting blood glucose specimen. Have the patient drink the dose of glucose solution required by the procedure.Collect blood at standard timed intervals. Blood, or blood and urine specimens, are then checked for glucose levels. The patient should be instructed to remain in the facility and remain seated between blood collections. The phlebotomist should check on the patient periodically between blood collections, especially during the first hour. For some patients, the glucose solution may cause nausea and vomiting and the test may need to be terminated.

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Standard and Post Prandial Glucose Tolerance Testing

Screening for and diagnosis of diabetes may require a patient to drink a specified dose of glucose in water ( 50 gram, 75 gram, or 100 gram), or eat a prescribed meal, depending on the test that is given. It is critical to the accurate interpretation of the test results that the correct procedure is followed. If you are responsible for administering the glucose dose and/or collecting the blood specimens, make sure that you follow the procedural steps as required by your laboratory's specimen collection manual, including:Verification of proper patient preparation (eg, when patient last had food or drink)Administration of correct glucose doseCollection of blood specimens at the correct time in relation to glucose administration or meal consumption

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Test for Gestational Diabetes

About 2 - 3% of pregnant women will develop gestational diabetes. Since women with gestational diabetes have a higher risk of losing their baby or having a baby with malformations, diagnosis and treatment of gestational diabetes is important.All pregnant women are screened for gestational diabetes at 28 weeks gestation using a modified glucose tolerance test. A fasting blood glucose is collected and then the patient drinks a 50-gram dose of glucose solution. A blood glucose specimen is collected one hour later.If the glucose results of the screen are abnormal, a 3-hour glucose tolerance test may be required. A fasting blood glucose is collected. The patient then drinks a 100-gram dose of glucose solution. Blood specimens are collected at one, two, and three hours after consumption of the glucose beverage. Be sure to label tubes as fasting, one-hour, two-hour, and three-hour. You may also be required to put the exact time of collection on the tube label.

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Case Study Three

Stop and Think ! A pregnant woman who is 28 weeks gestation has come to the blood collection area for a three-hour oral glucose tolerance test to confirm a diagnosis of gestational diabetes. The procedure requires the collection of a fasting blood sample followed by administration of a 100-gram load of glucose, which is administered in an orange-flavored beverage. Blood specimens will then be collected at one, two, and three hours. The woman has finished drinking the beverage, which she had a difficult time finishing, and you instruct her to have a seat in the waiting room until you come get her to have the one-hour post-glucose blood sample collected. After 30 minutes, she comes to tell you that she has just vomited.Consider how you would handle this or a similar situation before proceeding to read the suggested solution on the following page.

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Case Study Three: Discussion

Case study: A pregnant woman who is 28 weeks gestation has come to the blood collection area for a three-hour oral glucose tolerance test to confirm a diagnosis of gestational diabetes. The procedure requires the collection of a fasting blood sample followed by administration of a 100-gram load of glucose, which is administered in an orange-flavored beverage. Blood specimens will then be collected at one, two, and three hours. The woman has finished drinking the beverage, which she had a difficult time finishing, and you instruct her to have a seat in the waiting room until you come get her to have the one-hour post-glucose blood sample collected. After 30 minutes, she comes to tell you that she has just vomited.Suggested plan of action:The best thing to do in this situation is to contact the patient's physician. The test may need to be terminated. It may not be appropriate to continue the test under these circumstances as the glucose test results may not be accurate. The physician would need to make this determination.

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Collection and Communication

The laboratory plays an important role in monitoring the level of therapeutic drugs. Communication with clinical personnel is critical. Blood specimens are collected at specific time intervals to determine the trough level and peak levels of the drug. The pharmacist uses these trough and peak values to adjust the dose of the drugs appropriately.It is the responsibility of the phlebotomist to obtain the specimen at the precise time ordered for the specific peak or trough drug level. With some drugs, altering the draw time by even 15 minutes can have an adverse affect on adjusting and administering the next drug dose.Obtain the specimen at the requested time. If the time is missed, ask the clinical staff if the test should still be obtained or if another draw time is desired. If the clinical staff still wants a specimen collected, make a note of the time the drug was administered in relation to when the specimen was collected.Failure to communicate could have an adverse effect on the patient who may be given too little or too much medication based on an erroneous test result.

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Collection From a Line

An arterial line or vascular access device (VAD) is used to provide easy access to a patient's circulatory system for administration of fluids and medications. Occasionally, blood specimens are drawn from a VAD, but phlebotomists are not usually authorized to collect these specimens. However, phlebotomists may sometimes assist when a clinical person is collecting blood from a VAD. If you are present, be certain that the person performing the collection flushes the line with at least 5 mL of saline and the first 5 mL of blood is discarded before collecting the sample. If this procedure is not followed, the specimen may be contaminated with heparin that was used to flush the line or be diluted. This could cause inaccurate test results.

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Intravenous Line

Blood specimens should not be collected from an arm into which intravenous (IV) fluid is being administered. If at all possible, the phlebotomist should draw blood from the opposite arm or hand. If an IV line is delivering fluid into the patient's vein and the specimen is drawn from that vein, the specimen may be contaminated and diluted by the IV fluid; the blood test results could then be erroneous.If the arm or hand opposite of the arm that contains the IV line is not accessible or cannot be used for another reason, a capillary collection may be an option, if only a small amount of specimen is needed. However, if a venipuncture is necessary and the arm that has the IV line in place is the only option, ask the clinical person in charge of the patient's care to turn off the patient's IV. Ensure that the fluid has stopped flowing through the line, and wait at least two minutes before performing the venipuncture. It is imperative that the phlebotomist witness that the IV has physically been turned off by the health care provider and then turned back on after the draw has been completed. A phlebotomist must not turn the IV on or off.

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Transfusion Reactions
Additional Testing

If preliminary testing suggests hemolysis or if the results are misleading, additional testing may be required. If human error has been ruled out during the clerical check, repeat ABO/Rh testing should be performed on the unit of blood or its segment and the pretransfusion sample to detect any sample mix ups and clerical errors. Antibody detection studies should be performed on the pre- and post-transfusion samples to look for any unidentified antibodies. If an antibody is identified, the donor cells should be tested for the corresponding antigen. The crossmatch should be repeated with pre-and post-tranfusion specimens using the indirect antiglobulin test (IAT). An incompatible crossmatch with the pretransfusion sample indicates an original error, either clerical or technical. Incompatibility with only the post-transfusion sample indicates a possible anamnestic response, as in a delayed hemolytic transfusion reaction (DHTR), or sample misidentification. The patient's first voided urine specimen should be examined for the presence of free hemoglobin. The patient's bilirubin levels may also be evaluated. A change from normal pale yellow serum to a post-transfusion bright or deep yellow serum should prompt an investigation for hemolysis. The maximum concentration of bilirubin following hemolysis is not usually detectable until 3 to 6 hours after transfusion. The hemoglobin and hematocrit can be tested to detect a drop in hemoglobin or failure of the hemoglobin to rise after transfusion. Important information about physical or chemical hemolysis may be gained from examining the returned unit bag. If hemolysis is present in the bag or tubing, a process that affected the blood should be suspected, such as inappropriate warming or a faulty infusion pump. If bacterial contamination is suspected, the unit should be cultured. A positive culture indicates a reaction due to bacterial contamination.

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