Alcohol Information and Courses from MediaLab, Inc.

Important safety precautions must be observed when handling cerebrospinal fluid. The following guidelines apply:Semi-automatic micropipettes and disposable plastic chambers are the safest option for CSF testing. Many laboratories still use the hemacytometer with disposable pipets.If disposable materials are not used, soak contaminated reusable pipets, hemacytometer and coverslip in 70% alcohol or Wexide.All disposable items should be placed in a biohazard container for appropriate disposal.Wash hands thoroughly when the examination is completed.Spinal fluids which are to be discarded must be placed in biohazard containers for appropriate disposal.Careful attention to specimen processing and handling will help ensure that accurate results are obtained.

Procedural Step Comment Caution Greet and positively identify patient Always use at least two patient identifiers to ensure positive patient identification. Never rely on name placards that are placed on or near the patient's bed or crib to identify the patient. If there is a discrepancy in identification, do not proceed until the discrepancy is resolved. Explain the procedure If the patient is a small child, be at eye level when explaining the procedure. Also explain the procedure to the parent(s). If the patient is aware of what will be happening there is less chance of the patient suddenly jerking away his/her hand when the puncture occurs. Position patient appropriately An outpatient who is a small child should sit on the parent's lap. If necessary, seek assistance for finger puncture if the patient is a small child. Cleanse hands and put on gloves Use soap and water or alcohol-based gel to cleanse hands. Cleanse hands before donning gloves and after removing gloves. Warm puncture site if needed Use the method that is approved by the laboratory for prewarming the puncture site. Never use a moist cloth that has been heated in a microwave as this may cause injury to the patient. Gather appropriate equipment Only have needed equipment at hand. Keep track of ALL equipment to prevent patient injury. Cleanse the puncture site Use 70% isopropanol unless the patient is sensitive to alcohol. Allow the site to air dry. Performing the puncture before the alcohol has dried may hemolyze the blood specimen. Securely grasp and puncture finger Puncture the side edge of the fleshy pad of fingertip. Avoid extreme side and tip of finger. Discard puncture device into appropriate container Puncture device should be discarded into a sharps container that is puncture-proof, has rigid sides, and has a lid Do not discard puncture devices into regular trash or biohazard bags. Injury to personnel who handle these bags may occur. Wipe away the first drop of blood Use slight pressure to facilitate blood flow. The first drop of blood contains tissure fluid that may contaminate or dilute the blood specimen and affect test results. Collect blood into container Allow blood to flow freely into the collection device. Tap the container gently on a hard surface to move blood further down into the tube if necessary. Do not "milk" the finger or scrape the collection device across the finger to obtain specimen; both actions may cause the specimen to hemolyze. Mix specimen immediately upon completion of the collection. Apply pressure to the puncture site to stop the bleeding. Use gauze to apply pressure to the puncture site. It is not advisable to apply an adhesive bandage over the skin puncture site if the child is less than two years old as the child may place the bandage in his/her mouth. Label specimen Specimen must be labeled in the presence of the patient. Unlabeled specimens will be rejected by the laboratory.

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.

Lead may be found on surfaces touched by children and adults. Lead may be present in the paint that was used in older homes or apartments, and it has even been detected in the paint used on some toys.Elevated lead levels in children can cause developmental delays. Many state governments closely monitor the presence of lead in children. To accomplish this, government agencies require official forms be completed and submitted for each patient at the time of specimen collection for lead testing. It is the responsibility of both the phlebotomist and healthcare provider to submit the completed form with the specimen. If an elevated lead level is obtained, the government authority can then track and monitor follow-up treatment for the patient. When the phlebotomist determines that a capillary puncture on the finger will be used to collect a specimen for lead testing, it is imperative that the patient's hands be washed with soap and water prior to the start of the collection to ensure the skin is free of any contaminant that could falsely elevate the test result. The patient should thoroughly wash his or her hands or if the patient is a child, the parent or guardian could be asked to assist the child. If necessary, wash the patient's hands yourself.It is important to note that washing hands with soap and water aids in removing surface lead but is not a substitute for the cleaning step in the blood collection procedure. The finger must still be cleansed with alcohol and allowed to dry before a dermal puncture is made.

We have listed the 'classic' cardiovascular risk markers as LDL-C, HDL-C and triglycerides. But there are many more cardiovascular risk markers as well as cardiovascular risk factors. A cardiovascular risk factor is a condition (not a laboratory analyte) that is associated with an increased risk of developing cardiovascular disease. Examples include: Age Gender (males are at increased risk) Heredity Hypertension Cigarette Smoking Obesity Diabetes StressThere are also negative risk factors, factors which decrease a person's risk of cardiovascular disease. Examples include: Optimal HDL-C concentration Exercise Estrogen Moderate alcohol intakeThis course will not focus on cardiovascular risk factors. Instead we will focus on newer, emerging cardiovascular risk markers. There are well over twenty well-studied cardiovascular risk markers; in this course we will focus on some of the more established markers and the ones which are becoming more commonly measured in the clinical laboratory. These include apolipoprotein A1/apolipoprotein B100, Lp(a), oxidized LDL, LpPLA2, hsCRP and lipoprotein particle size and concentration.It is important to remember that the association between a cardiovascular risk marker and actually having or developing cardiovascular disease is a statistical one. The fact that a patient has a particular risk marker which is abnormal simply increases the probability of developing cardiovascular disease, it does not mean that he or she is certain to develop cardiovascular disease. Conversely, if an individual does not have a particular cardiovascular risk marker present it does not guarantee protection against cardiovascular disease. We must always remember that some percentage of individuals who have heart attacks or strokes will not have abnormal risk markers present.

Deferoxamine (DFO), an iron chelating agent, may be used to reduce iron overload in patients for whom phlebotomy is contraindicated or not well tolerated. Examples include patients with sickle cell disease or thalassemia whose anemia would be exacerbated by phlebotomies. DFO is seldom used to treat hereditary hemochromatosis (HH) due to the low cost and efficacy of phlebotomy therapy. DFO is typically administered by intravenous or subcutaneous infusion.Patients with HH may be counseled to avoid alcohol use in order to avoid liver damage. With the exception of iron supplements, dietary restrictions on iron ingestion are rarely advised.

Sample Type Required: Deparaffinized and re-hydrated tissue sections on positively charged slides. Fixative: 10% Neutral Buffered FormalinStepReagentTimeTechnical Notes1Weigert's Iron Hematoxylin Working Solution½ Solution A (Hematoxylin and 95% Alcohol) and ½ Solution B (Distilled Water, Hydrochloric Acid and 29% Ferric Chloride Solution)7 MinutesBe careful not to overstain with Hematoxylin as this can obscure the carminophilic tissue elements.If Gills hematoxylin is used in place of Weigert's Iron Hematoxylin the mucin may have a bluish cast.2Running Tap Water10 Minutes3Working Mucicarmine Solution10mL Mucicarmine Stock Solution and 40mL Distilled Water60 MinutesStock Mucicarmine should be stored refrigerated to slow down deterioration of the solution. Expiration of the solution should be closely monitored. Do not freeze solution to avoid frequent freeze-thaw cycles that may cause deterioration.4Distilled Water1-2 Changes5Metanil Yellow0.25% Solution30 Seconds to 1 MinuteBe careful no to overstain with Metanil Yellow as this can obscure carminophilic tissue elements. Tartrazine can be used in place of Metanil Yellow. Expected ResultsMucin = Deep Rose Red Capsule of Cryptococcus = Deep Rose Red Nuclei = Black Other tissue elements = Blue or yellowPost Staining Procedure: Tissue sections should be rinsed well in distilled water, dehydrated with 95% and absolute alcohols, cleared and cover-slipped. Mucicarmine stock solution should be stored refrigerated Room temperature storage will accelerate the chemical breakdown of the solution. Frozen storage is not recommended to avoid repeated freeze-thaw cycles that may also affect the chemical reactivity of the stain.

The PTAH staining method relies on acid-base chemistry to stain collagen and muscle fibers. These fibers are demonstrated using a tungsten mordant provided by the phosphotungstic acid. This mordant binds hematein and stains selective tissue components blue, while the phosphotungstic acid is believed to stain other tissue components a red-brown color. Tissue should preferably be fixed in Zenkers, which is believed to intensify staining reactions. However, tissues fixed in formalin are commonly post-fixed in Zenker’s solution for this purpose. A section of skeletal muscle tissue can be used for quality control.The amount of phosphotungstic acid in the staining solution is far greater than the amount of hematein and it is believed that tungsten binds all available components blue, while the phosphotungstic acid is thought to stain the red-brown components. This stain has been referred to as a polychrome stain because one solution gives two major colors. The components colored red-brown will lose this color with water or prolonged alcohol washes. Therefore, dehydration of the tissue section following staining MUST be rapid. (Ref4)

This slide shows a marrow aspiration smear with numerous ring sideroblasts. Normal red cell precursors have only one or at most two granules of iron in their cytoplasm. These abnormal red cell precursors have numerous iron containing granules in their cytoplasm indicating abnormal iron incorporation. This iron is actually incorporated into mitochondria. Ring sideroblasts can be seen in idiopathic sideroblastic anemia, and in sideroblastic anemia induced by drugs, lead poisoning, and alcohol abuse.

An individual whose license that has been put on probation for violating the laws of the Board may be subject to any or all of the following requirements, as assigned by the Board: Practice only under direct supervision of a licensed clinical laboratory personBe reviewed on a quarterly basis by his / her supervisor, with reports submitted to the BoardSubmit a personal quarterly report to the Board describing the individual's progressComplete additional continuing education requirementsConsult a psychiatristNot consume alcohol or use any controlled or illegal substancesAttend AA or NA meetings weeklyUndergo and pay for random drug testingPay an administrative fineFailure to comply with all conditions of the probation will mean that the individual's license will be suspended or revoked.

Standardized systems should be used in virtually every circumstance to reduce errors. For example, medical errors can be avoided by using the standardizing procedure for preparing a venipunture site before drawing a blood alcohol specimen. A standardized system for this procedure is developed, published, trained, and posted. Everyone learns one protocol. Encouraging them to review and use the procedure for drawing blood alcohol tests avoids the error of using alcohol wipes to prepare the venipuncture site.

Alcohol-based antiseptic hand cleanser can be used in place of soap and water. Place a dose of the antiseptic hand cleanser into the palm of your hand and rub your hands together, spreading the solution thoroughly over both hands, particularly around nail beds and under jewelry, until the hands are dry.

The intent of the safety data sheet (SDS) is to answer these questions:What is the material?What information needs to be known immediately?What should be done in cases of emergency situations?How can hazardous situations be prevented from occurring?What other useful information is there on this material?As of June 1, 2015, new SDSs supplied by the chemical manufacturers must be in a uniform format that includes 16 specific sections. The SDS will include the section numbers, the headings, and associated information under each heading. The 16 section headings are:IdentificationHazard identificationComposition/information on ingredientsFirst-aid measuresFire-fighting measuresAccidental release measuresHandling and storageExposure controls/personal protectionPhysical and chemical propertiesStability and reactivityToxicological informationEcological informationDisposal considerationsTransport informationRegulatory informationOther information (includes the date of preparation or last revision)

Laboratory specimens that are unlikely to cause disease and do not meet the criteria for category A or B substances are not subject to Division 6.2 regulations. Specimens for which the hazardous materials regulation (HMR) does not apply include human or animal samples (including, but not limited to, secreta, excreta, blood and its components, tissue and tissue fluids, and body parts) being transported for routine testing not related to the diagnosis of an infectious disease. This includes specimens that are being sent for:drug or alcohol testing cholesterol testing blood glucose level testing prostate specific antibody (PSA) testing testing to monitor kidney or liver function pregnancy testing tests for diagnosis of non-infectious diseases such as cancer biopsies The US Department of Transportation (DOT) has no "Exempt Specimen" classification and there are no DOT guidelines for packaging non-regulated specimens.* According to the DOT, in the U.S., if a package is marked as "Exempt Human/Animal Specimen" the understanding is that it contains no infectious substance. However, both IATA and the US Postal Service (USPS) have these requirements for packaging exempt specimens: Packaging IssueIATAUSPSType of packaging requiredTriple packagingTriple packagingOuter containerOne dimension must be a minimum of 100 mm X 100 mm (approximately 4 x 4 inches) Must be able to survive a drop test of 4 feet One dimension must be a minimum of 100 mm X 100 mm (approximately 4 x 4 inches) Must be able to survive a drop test of 4 feet Quantity limits: outer containerNone NoneQuantity limits: Primary receptacleNone500 mLQuantity limits: secondary packagingNone500 mL* Non-regulated specimens may become regulated because of preservatives, such as 10% formaldehyde (class 9) or 25% formaldehyde (class 8); or 25% ethanol (class 3).

The following is a list of enzymes for which known mutations have been associated with clinical effects. Enzymes Substrates (Drugs) Acetylaldehyde dehydrogenase Alcohol Acetylcholinesterase Succinylcholine Alcohol dehydrogenase Alcohol Dihydropyrimidine dehydrogenase Fluorouracil CYP2C9 Warfarin, phenytoin, losartan CYP2C19 Diazepam, omeprazole (Prilosec) CYP2D6 Many antidepressants, opioids, antiarrhythmics Glucose-6-phosphate dehydrogenase Aspirin, quinidine N-acetyltransferase Procainamide, isoniazid Thioprine methyltransferase 6-mercaptopurine UDP-glucuronosyl transferase Acetaminophen, tolbutamide, irinotecan

Marcie Moore was a phlebotomist at a community hospital in Atlanta. It was her week to collect the pediatric unit and she was on her way to the room of a newborn for which she had just received orders to draw a STAT BMP (chem-7) and bilirubin. After informing the mother of the baby about the test she needed to perform, Marcie set up to perform a heel stick on the baby. Marcie chose a site on the outer edge of the heel on the bottom of the baby's foot ( the correct area for a heel stick) and made a small incision with a Tenderfoot lancet after cleaning the site well with alcohol.She immediately began collecting the blood in the correct tube for the BMP and bilirubin. Blood flow was not strong so Marcie squeezed the baby's foot a little to help the blood come out faster – the newborn was screaming and Marcie could tell it was making the mother uncomfortable. She wanted to hurry and get done so the mother could hold the baby.After the chemistry tech ran the blood tests on the tube, she informed Marcie that the newborn had a panic potassium level which did not coincide with the previous blood work on the newborn. Also the chemistry instrument could not perform the bilirubin due to hemolysis. Marcie was asked to recollect the specimen.

During a finger stick procedure it is important that the lancet be positioned on the finger so that the incision is perpendicular to the fingerprint. This allows a larger amount of blood to flow. It is also important to wipe away the first drop of blood that emerges form the incision with clean gauze, since it may contain tissue fluids that can cause incorrect test results. The first drop of blood may also contain traces of alcohol remaining from the cleaning step. Alcohol may break up or hemolyze blood cells, causing incorrect results.Relevant topics:Finger-stick collections, Finger-stick: site preparation, Finger-stick: puncture, Wipe away the first drop, Finger-stick specimen collection

A phlebotomist from the laboratory at Midtown Memorial Hospital was working evening shift. Her shift ended at 11 PM and it was 10:30 PM. She suddenly got orders for a STAT blood culture on the second floor. The order specified blood culture times two, 30 minutes apart. The phlebotomist went to the patient's room and decided to collect both blood cultures at the same time form the same site so she would be able to leave on time without having to come back in thirty minutes to collect the second set. She also wanted to "save" the patient from an extra stick. While the phlebotomist was preparing for the collection, she realized she didn't have any Betadine on her tray, and decided she would just clean the site twice with alcohol. She finished the blood culture collections and was able to leave by 11 PM.

Basic equipment includes: Alcohol swab, Bandage, Tube(s), Needle, Needle holderDo not remove the needle cover until you are ready to perform the venipuncture.

Allow the alcohol to dry prior to performing the venipunctureDrying gives the alcohol time to disinfect the site.It also tends to prevent a burning sensation from occurring during venipuncture.

Puncture the left or right side (outskirt) of the heel, not the bottom of the foot.Wipe away the first drop of blood since it may contain excess tissue fluid or alcohol which could alter test results.

Clean the site with an alcohol prep pad and allow it to dry.

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.

Clean blood culture bottles while the iodine on the venipuncture site is drying. Wipe the tops of the blood culture bottles, first with a new iodine swab, then with a clean alcohol pad.

Collect the blood specimen next, if required.Be sure to use the iodine swab provided in the collection kit to disinfect the venipuncture site.Do not use an alcohol swab, as this might lead to suspicion of a falsely elevated blood alcohol result.

Preanalytical Error What is it? How does it happen? What is the result? Hemolysis Red blood cells (RBCs) break and release contents of cell into plasma. Needle incorrectly positioned in vein; cells forced to squeeze through opening. Needle gauge too small; slow blood return into tube. Vigorous mixing or shaking of tube. Alcohol on skin that has not had sufficient time to dry. Some test results may be falsely elevated. (Potassium is especially affected by hemolysis.) Patient may have to be re-drawn. Clotted specimen Clumped or clotted cells in specimen that requires anticoagulated or whole blood Insufficient mixing of blood with anticoagulant in tube. Delay in mixing tube. Slow filling tube. Inaccurate test results for cell counts and clotting studies. Patient may have to be re-drawn. Tube filled to incorrect volume Too little or too much blood in tube. Tube removed from needle too quickly. Vacuum in tube has been compromised due to use of tube past the expiration date (Results in a short fill). Manual fill of tube may lead to over-fill. Test results may be unreliable due to dilution errors. Patient may have to be re-drawn.

A tourniquet is used by the phlebotomist to assess and determine the location of a suitable vein for venipuncture. Single-use, latex-free tourniquets are preferred but reusable tourniquets are acceptable. However, if the reusable tourniquet becomes contaminated with blood or body fluid, it must be discarded immediately to avoid the spread of harmful contaminants to other patients. Follow the guidelines established by your facility for cleaning reusable tourniquets.Proper application of a tourniquet will partially impede venous blood flow back toward the heart and cause the blood to temporarily pool in the vein so the vein is more prominent and the blood is more easily obtained. The tourniquet is applied three to four inches above the needle insertion point and should remain in place no longer than one minute to prevent hemoconcentration. If the tourniquet is used during preliminary vein selection, it is best to release the tourniquet after assessing the vein and while you are assembling your supplies. Reapply the tourniquet just before starting the venipuncture; it should then be released soon after the needle has been inserted into the vein and the blood flows into the first tube. If collecting multiple tubes, the tourniquet may remain in place until blood enters the last tube.

Safety precautions should be observed when handling seminal fluid. The following guidelines should be followed:If non-disposable items are used, soak contaminated items(e.g., hemocytometers and coverslips) in 70% alcohol or other appropriate decontaminate.All disposable items should be placed in a biohazard bag.Non-latex or powder-free latex disposable gloves must be worn and hands thoroughly washed when the examination is completed.Seminal fluids that are to be discarded should be placed in biohazard bags.

During a blood collection, bacteria that is present on the skin surface may adhere to the outside of the needle as it enters into the vein. This can allow bacteria to infect the puncture site. A serious infection of the blood (septicemia) or of the tissue (cellulitis) may result. To avoid an infection, it is imperative that the phlebotomist uses a technique that thoroughly cleanses the skin at the site prior to venipuncture.Once the phlebotomist locates a suitable vein for venipuncture, the site of the vein that will be punctured is cleaned with a pre-packaged wipe saturated with 70% isopropyl alcohol.The site is cleansed using a "target" motion beginning at the center of the site and moving outward in concentric circles applying enough pressure to move surface bacteria away from the puncture point. (This is demonstrated in the image on the right). It is not recommended to use a scrubbing back and forth motion to clean the site since you may drag bacteria from a dirty area back into the clean area. Allow alcohol to air dry for effective disinfection of the site. Never use non-sterile gauze to wipe dry the alcohol as this will contaminate the site.During the remainder of the procedure, the site must NOT be touched by anything that has not been cleaned in an identical manner. The phlebotomist should avoid retouching the site after cleaning. If it is absolutely necessary to re-palpate, the phlebotomist MUST clean the gloved finger in a manner identical to the above procedure. Make certain that no other piece of equipment touches the site. This includes ends of the tourniquet and gauze. If you suspect that your needle has touched the site before entry, dispose of the needle, re-clean the site and repeat the procedure using a new needle. If a patient complains that there is redness or pain at the puncture site, even hours or days after the procedure, immediately refer the patient to his/her physician for evaluation.

Influenza A viruses, including the 2009 H1N1 strain, are able to survive and maintain infectivity on surfaces for extended lengths of time. Influenza A viruses typically remain infectious for 12 - 48 hours on non-porous surfaces, for 8 - 12 hours on cloth or paper, and for 5 minutes on hands.To reduce spread of the virus from person to person, it is important to discard contaminated items such as tissues and laboratory testing supplies and to wash hands frequently. To eliminate viruses from contaminated surfaces, a number of disinfectants can be used, such as chlorine, hydrogen peroxide, detergents, iodophores, and alcohols. Influenza viruses also can be rapidly inactivated with heat from 167 - 212°F.

Leucine crystals indicate a problem with the metabolism of the amino acid leucine. These crystals are round to oval with radiating bands going from a center point out to the periphery, often referred to as a "wagon wheel." These crystals are soluble in hot alcohol and alkali.

As mentioned earlier, differentiation is used with regressive staining techniques and is used to remove the excess dye that has been taken up into the tissue until a crisp delineation of the desired parts is achieved.Basic dyes are differentiated using a weakly acidic solution prepared with alcohol.Acid dyes are differentiated using a weakly basic solution prepared with alcohol.Aqueous based differentiating solutions are not recommended as the dyes will diffuse more rapidly and over-differentiating becomes a problem.

Eosin is any one of a class of rose-colored stains or dyes and many types can be obtained commercially. Some of the more common ones are: Eosin Eosin Y (most widely used and is soluble in both alcohol and water) Alcoholic eosin Y Eosin B Eosin- phloxine Picro-eosin

When there is a need to bypass the lengthy routine processing steps for tissue, as in cases where an urgent diagnosis is needed, frozen sections can be prepared. The tissue is cut on a cryostat, which is basically a microtome enclosed in a deep-freeze compartment. The ice surrounding the tissue takes the place of paraffin as the embedding medium; therefore, the and hydration step can be eliminated when staining the slides. Sample rapid H&E stain for frozen sections:1. Formol-acetic alcohol (quick fixative) for 20 seconds2. Water rinse3. Harris's Hematoxylin for 1 1/2 minutes4. Water rinse5. Ammonia water - dip until section turns blue6. Water rinse7. Eosin for 10 seconds8. Water rinse 9. Dehydrate, clear, coverslip

When the slides go from the xylene in the deparaffinizing step to the first alcohol in the hydration sequence, the sections should turn slightly opaque. If there are clear patches present then the deparaffinization was incomplete. Possible Causes: Water still on the slides after the drying oven Xylene contaminated with waterRemedies: Remove excess water by putting the slides back in the oven or into 100% alcohol Change reagents regularly to ensure that the xylenes stay water-free Drain slides well before transferring them to the next reagent

Differentiation of the eosin occurs best in 70% alcohol and to a lesser extent in the higher grade alcohols; therefore, adding a 70% alcohol to the dehydration series or adjusting the time in the 70% alcohol will aid in obtaining the desirable three distinct shades of eosin. The pH of the eosin is another possible cause of poor differentiation. Carry-over of the alkaline bluing solution into the eosin can occur if the slides are not rinsed adequately prior to the eosin. This increase in pH will interfere with the uptake of the dye into the tissue. Proper rinse time, adjusting the pH with the addition of acetic acid, and/or regular changes of fresh eosin will resolve this problem.The image on the left is one shade of pale pink as opposed to three shades of pink in the image to the right. This was probably due to being over-differentiated in the alcohols.