Sterile Information and Courses from MediaLab, Inc.

Under normal circumstances, fluids collected from any enclosed body space such as a cerebrospinal fluid (CSF), pleural, peritoneal, pericardial or synovial fluids, should be sterile. When an infection occurs within a body cavity, the fluid that is collected from that site for diagnostic purposes will potentially have infectious organisms present on the cytospin. There can be several types of organisms demonstrated on the Wright stained preparation.Bacteria and fungus are the most common but it is possible to demonstrate the presence of protozoan parasites as well.Spirochetes and acid fast bacilli (AFB) such as mycobacteria will not stain with Wright stain so they will not be detected even if they are present.Since bronchial alveolar lavages, or BALs, are collected through an open airway it is normal to observe respiratory flora, however yeast and hyphae are never normal in a BAL.It is standard practice before reporting the presence of bacteria in a fluid to correlate/confirm the findings with the microbiology lab.

When the technologist accompanies the clinician to assist with the bone marrow aspiration procedure to make smears at the bedside, it is necessary to understand the role of the clinician and the technologist.The clinician is responsible for patient positioning and sterile preparation, pain control, and performing the aspirate and biopsy. The clinician often hands off sample syringes to the technologist, once collected. The clinicians are responsible for providing the procedure kit and fixative for the biopsy, all labels, and obtaining the requisitions and a copy of the clinical history for the hematopathologist. The technologist will set up a mini workspace near the bedside where the samples are split into the required tubes. Smears are then prepared from the aspirate as well as biopsy touchpreps before the biopsy is placed in fixative. In this setting the technologist will usually deliver the samples and requisitions to pathology and continue the processing procedure.The kit the technologist brings to the bedside usually contains mini petri dishes, coverslips, slides, microcapilary tubes or Pasteur pipettes, micro-pipette bulb and the various evacuated blood collection tubes and media flasks required for the standard bone marrow draw.Most institutions will have a standard draw and testing protocol designed to ensure that enough sample is obtained to cover all of the usual testing requirements. An example would be a three-syringe-draw with the first two syringes containing no anticoagulant and the third syringe rinsed with preservative-free heparin. The first dry pull would be split between a green and a purple top evacuated blood collection tube and would be used for morphology (EDTA) and flow cytometry and cytogenetics (green) if needed. The second dry pull is split into two additional purple top tubes plus a green top tube and would be used for molecular assays such as SNP array, Flt-3, JAK2, MPL mutation, etc. The final heparinized syringe could be used for other treatment protocol requirements or to provide sample for additional assays.

Citron DM. Appelbaum PC.: How far should a clinical laboratory go in identifying anaerobic isolates, and who should pay? Clinical Infectious Diseases. 16 Suppl 4:S435-8, 1993 Identification of anaerobic bacteria in specimens from sites of infection due to mixed organisms can be time-consuming and expensive. Laboratories should limit anaerobic workups by testing only those specimens that have been properly collected and transported to the laboratory. Use of selective and differential media for initial processing can provide rapid and relevant information to the clinician. Anaerobes isolated from normally sterile sites and sites of serious infection should always be completely identified. Group-or genus-level identifications may suffice in other instances. The Bacteroides fragilis group of organisms should always be identified because of their virulence and resistance to many antimicrobial agents. Some of the other organisms that warrant identification include Clostridium septicum (associated with gastrointestinal malignancy); Clostridium ramosum, Clostridium innocuum, and Clostridium clostridioforme (which are resistant to antibiotics); Clostridium perfringens (a cause of myonecrosis and gas gangrene,potentially serious infection); anaerobic cocci (which may be resistant to metronidazole and clindamycin); and fusobacteria (which may be virulent and resistant to clindamycin and penicillin).

The cerebrospinal fluid sample is obtained by a physician usually via lumbar puncture in the L3-L4 region. The opening pressure is first measured (nl 90-180 mm of water in lateral position) and if it is elevated greater than 200 mm, no more than 2 ml of CSF should be withdrawn. Sterile technique is always used to reduce the risk of infection. Care must be taken to avoid injury to neural tissue.

In addition to the puncture device, additional equipment may be required when performing a successful dermal puncture.Plastic microcollection devices: Plastic microcollection devices are small plastic tubes designed to collect capillary blood from a dermal puncture wound. Each small collection tube is color-coded in the same manner as blood collection tubes used for venipuncture. The color of the cap of each container tube corresponds to the type of additive inside the tube, most often an anticoagulant. The additive coats the inside of the tube. Examples of microcollection devices are shown below. Heel warmer: It is best practice to warm the heel of an infant with a warming device known as a heel warmer. The heel warmer, when activated, is designed to warm its contents to a standardized temperature. This temperature will be hot enough to effectively warm the heel and facilitate blood flow to the area without causing heat injury to the patient. It is unacceptable to warm a cloth using a microwave. There may be "hot spots" on the cloth that could potentially burn the patient. Keep in mind, what may feel warm to you, the phlebotomist, may feel hot to your patient!Plastic or Mylar-wrapped capillary tube: In some facilities blood from a capillary puncture is collected directly into a capillary tube. These tubes are very delicate and must be used with great caution. As soon as the tube is two thirds to three-fourths filled, one end is sealed to prevent blood from leaking out.Glass microscope slides: In some facilities, the person collecting the capillary specimen may also be required to prepare a blood smear for laboratory examination. A drop of blood is placed directly on a glass slide and spread to create an area for cell examination. If you are required to prepare blood smears, remember that the slide is considered infectious until fixed or stained. It is also important to remember that glass is a sharps hazard. If not used correctly, the glass may cause injury to both the patient and the phlebotomist. Be as cautious with a glass slide containing blood as you are with a contaminated needle. Dispose of glass slides that will not be used for testing in approved sharps containers.Alcohol and gauze pads: Alcohol is the disinfectant of choice for dermal puncture. The alcohol must be allowed to air dry, which will prevent hemolysis of the specimen and discomfort for the patient. A piece of clean or sterile gauze is used to wipe away the first drop of blood. Gauze is also used to apply pressure to the wound after the specimen collection is complete to stop the wound from bleeding.Iodine or other approved cleaning agents may be used as an alternative to alcohol.Bandage: It may be necessary to apply a bandage to the puncture wound on a finger or heel if the site continues to bleed. However, it is NOT recommended to bandage the finger of a child who is 2-years-old or younger since the bandage may become a choking hazard if the child puts that finger in his/her mouth.Personal protective equipment (PPE): All healthcare professionals that may come in contact with blood and/or body fluids while performing a laboratory procedure are required to wear intact gloves. It is against safety guidelines to alter gloves in any way that may compromise the integrity of the gloves. Eye protection, such as safety goggles, is recommended if there is the possibility of a splash of blood while collecting a capillary blood specimen. In many facilities, special gowns are required in some patient areas such as special-care nurseries. Always follow the policies of your facility in regard to PPE.

Successful electrophoresis requires application of correct amount of sample with clean applicators and no air bubbles. Many gel electrophoresis for manual sample application include a sample application template to lay over the gel, a micropipet, or a thin-wire applicator. Polyacrylamide gels will have wells in the gel for the sample. Sterile pipet tips are required when applying DNA and RNA specimens.

Submerge the affected part in cold water for 10 to 45 minutes. This will relieve pain and cool tissues to prevent further damage.Give aspirin or ibuprofen to relieve pain and reduce inflammation.Cover second degree burns with a dry nonstick sterile dressing.

Give first aid. Wash needlesticks and cuts with soap and water. Flush splashes to the nose, mouth, or skin with water. Irrigate eyes with clean water, saline, or sterile irrigants. Report exposure to supervisor.

The physician ordering the bone marrow is responsible for providing information about the procedure to the patient or parent or guardian, if the patient is a child. In order to reduce the patient's anxiety about the procedure, the physician may prescribe a mild sedative to be administered about an hour before the bone marrow is scheduled. The site is aseptically prepared by shaving, if necessary, washing with soap and water, applying antiseptic and draping the area with sterile towels. A local anesthetic, such as 2% xylocaine, is injected into the bone, penetrating the covering of the bone called the periosteum. Since a number of nerve endings are located near the surface of the bone, it is important to be sure that this area is anesthetized.

Sample preparationSamples for flow cytometric analysis are prepared based on the following procedural steps. Prepare the sample according to cell type. Bone marrow (BM) and peripheral blood (PB): Prepare a blood smear, stain with Wright's stain, and scan under the microscope to identify basic cell distribution and morphology. BM can contain spicules; these samples need to be filtered. Tissue: Mince and filter tissues in a sterile cell culture media to release cellular components from the solid tissue form. Fluid: Filter fluid, prepare a cytospin, and scan under a microscope to identify cellular components. Obtain a white blood cell (WBC) count. Unless red blood cells are the population of interest, they should be lysed with a mild agent that will preserve the integrity of the cells targeted for analysis. If the red blood cells are not lysed, they lead to false analytic results. Adjust the WBC count by concentrating the WBCs to optimize for ‘staining' with monoclonal antibodies (MoAbs). Incubate the prepared cell concentration with assorted monoclonal antibody concentrations to allow antigen-antibody complexes to form. Lyse red blood cells (RBCs) with ammonium chloride or equivalent that will preserve cellular viability and integrity. The purpose is to eliminate RBCs while maintaining WBC integrity. If RBCs remain in the sample, they will interfere with the cell scatter plot and skew the results. Centrifuge to precipitate cells. Pour off supernatant. Wash in phosphate buffered saline (PBS) or equivalent to eliminate cellular debris and unbound MoAbs, centrifuge, and decant. Resuspend cells in PBS. Analyze cells using flow cytometer.

Staphylococci are non-motile, non-spore-forming, gram-positive organisms occurring singly, in pairs, tetrads or in clusters resembling grapes. More than 20 species have been identified; three species are significant in their interactions with humans - S. aureus, S. epidermidis and S. saprophyticus.The staphylococci are members of the normal flora of the skin and mucous membranes of humans and warm-blooded animals. Colonization of the nares (nostrils) and skin can provide large reservoirs of organisms for transmission. Approximately 25-30% of the general population are colonized by Staphylococcus aureus, mainly in the nasal passages, but the organism can be found in most anatomical sites including the skin, oral cavity and GI tract.Infections are frequently acquired when the colonizing strain gains access to a normally sterile site as a result of trauma or abrasion to skin or mucosal surface. S. aureus infections range from superficial, localized skin infections, such as folliculitis, to deeper, more serious skin lesions and the more serious toxin mediated conditions – scalded skin syndrome and toxic shock syndrome.

Bandaging materials include: Sterile gauze pads.Band-Aids.

After collection is completed, apply pressure to the puncture site with a sterile gauze pad until bleeding has stopped.Do not apply an adhesive bandage to an infant's foot since it may injure its delicate skin.

Blood is normally sterile. Any bacterial growth in the bloodstream is abnormal, and is an important cause of fever.Blood culture means the incubation of blood in appropriate media to allow growth and identification of bacteria or other organisms that may be present in a patient's bloodstream. Blood cultures are performed on febrile patients to identify and treat bloodborne organisms with the most appropriate antibiotic.

These items are needed to obtain a blood culture specimen :Gloves (sterile if available)Alcohol pads and sterile gauze padsTourniquet and iodine swabsBlood culture bottlesSyringes, needles, and/or evacuated tube system.

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.

Epithelial cells in large numbers within sputum smears means that the specimen is predominantly oral saliva, rather than true sputum from the lung. Epithelial cells in urine smears indicate that the sample has been contaminated by organisms found on the vulva or distal urethra. Bacteria found near or on epithelial cells are usually normal contaminating bacterial flora.White blood cells indicate inflammation and possible infection. The direct smear examination should focus within and around these cells.Red blood cells in a direct smear are not usually significant.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.Hyphae are more likely to indicate the presence of fungal infection, but this determination requires correlation with clinical findings.Bacteria found in spinal fluid, blood, tissue and specimens from other sterile sites are always significant.Body fluids which are normally sterile must be examined carefully. If only one organism per oil immersion field is identified, then there are about 105 organisms per mL present in the sample! 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.

Blood is normally sterile. Any bacteria in the bloodstream is abnormal. A blood culture is collected to detect the presence of bacteria in the bloodstream. Blood is collected into appropriate media to allow for growth and identification of bacteria or other organisms that may be in the patient's bloodstream. A blood culture set usually consists of two bottles: an aerobic bottle and an anaerobic bottle. Blood cultures are usually ordered in multiple sets drawn from separate sites at different times. An improperly collected blood culture can have a serious impact on the care and treatment of a patient. If bacteria enters the culture vial from sources other than the blood, as a result of improper specimen collection, a patient may needlessly be treated for an infection that is not present. On the other hand, some collection errors may cause negative culture results when the patient actually has bacteria in his/her blood. A false-negative culture result could be a life-threatening error.

The manufacturer's instructions (package insert) lists the acceptable respiratory specimen types for testing in each rapid influenza A/B test kit. The following specimen types are preferred for influenza virus diagnostic testing: nasopharyngeal swabs, washes and aspirates; endotracheal aspirates, and bronchoalveolar lavage (BAL). The CDC recommends nasopharyngeal washes for patients less than 3 years old and nasopharyngeal swabs for patients over 3 years of age. The image on the right illustrates the correct area to swab when collecting a nasopharyngeal specimen. Two swabs or washes would facilitate both rapid influenza A/B testing and a second specimen that could be sent for confirmatory H1N1 testing by RT-PCR if needed. Nylon fiber flocked swabs have been shown to be more effective than dacron or other woven fiber swabs for the collection of virus from the nasopharynx. The soft brush-like nylon fiber swabs are more efficient at collecting cells and viral particles and subsequently releasing virus when swabs are placed into viral transport media due to the capillary action of the flocked swab. It is suggested that the ill patient should have specimens collected as soon as possible after the onset of symptoms. For certain patients, more than one specimen may be necessary to confirm the presence of the virus. Nasopharyngeal swabs should be placed into sterile universal transport medium (UTM) or comparable viral transport media and promptly delivered to the laboratory within 2 hours. If processing is delayed longer than 2 hours, respiratory specimens should be placed in a refrigerator (held at 40C). Viruses usually remain stable for 2 to 3 days when held at this temperature.

A urine specimen for urinalysis should be collected in a clean, dry, disposable container. If the sample is to be cultured, the container must be sterile. The preferred method is the " midstream clean catch" collection. The external genitalia are cleansed with a mild antiseptic solution. The first part of the urine stream is discarded while collecting only the midstream portion of the urine.