# Which type of test tube should not be used for blood collection?

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The following question is presented in my biology textbook:

You are required to draw blood from patient and keep it in a test tube for analysis of blood corpuscles and plasma. You are provided with the following four types of test tubes. Which one of them will you not use for the purpose?

1. Test tube containing calcium bicarbonate
2. Chilled test tube
3. Test tube containing heparin
4. Test tube containing sodium oxalate

In thinking through the question, I reasoned that since we are collecting blood from a patient for biochemical assay or hematocrit, we would want it to be in an anticoagulated stage. Sodium oxalate and heparin are both anticoagulants and therefore should serve my purpose well. I don't think a chilled test tube would have any effect directly, except the fact that low temperatures delay clotting. So, I had chosen the test tube containing calcium bicarbonate to be unsuitable, as $$ce{Ca++}$$ is one of the factors required for clotting and hence would accelerate the clotting procedure.

The textbook disagrees with me, and gives (3) as the answer.

Is this an error in the textbook key, or a flaw in my reasoning? Which should be the correct answer, and why?

An unclotted blood sample needs something to prevent clotting. Extracellular calcium is required for both the coagulation cascade and platelet activation. It even has its own name in this context, Factor IV. This why EDTA, a calcium chelator, is used in some blood collection tubes to delay clotting.

So, as you say, the most correct answer would be (1): Test tube containing calcium bicarbonate. That is the opposite of the kind of tube you should use for this type of sample.

The answer given by the key, (3): Test tube containing heparin, is incorrect. Heparin tubes are a good choice for plasma and whole blood analysis, as you indicated. Heparin is a good anticoagulant, both in vitro and in vivo.

See this nicely laid out list from UCI medical school for an example of specimen requirements for specific tubes.

## Specimen Requirements/Containers

Laboratory test results are dependent on the quality of the specimen submitted. If there is any doubt or question regarding the type of specimen that should be collected, it is imperative that the laboratory is called to clarify the order and specimen requirements.

Most laboratory tests are performed on serum, anticoagulated plasma or whole blood. Please see the individual test directory listings for specific requirements.

Plasma: Plasma: Draw a sufficient amount of blood with the indicated anticoagulant to yield the necessary plasma volume. Gently mix the blood collection tube by inverting 8-10 times immediately after collection. The majority of samples require separation of plasma from cells within two hours of collection.  However, there are few tests that require separation within 15-30 minutes.  Please refer to our laboratory test directory for additional information. All specimens must be delivered to the laboratory within 4 hours of collection.

Serum: Draw a sufficient amount of blood to yield the necessary serum volume.  Invert tube 5-10 times to activate clotting. Allow blood to clot at room temperature for 30 minutes. NOTE: Avoid hemolysis.

Whole Blood:  Draw a sufficient amount of blood with the indicated anticoagulant. Gently mix the blood collection tube by inverting 8-10 times immediately after collection. NOTE: Tubes intended for whole blood analyses are not to be centrifuged and separated.

All patient specimens must be place in biohazard bags for transport to the laboratory.

Specimen Collection Tubes

• Gold-top serum separator tube (SST)
This tube contains a clot activator and serum gel separator – used for various chemistry, serology, and immunology tests. If the specimen requirement for a test is red-top tube(s), do not use gold-top/SST tube(s).
NOTE:
Invert the tube to activate the clotting let stand for 20-30 minutes before centrifuging for 10 minutes. If frozen serum is required, pour off serum into plastic vial and freeze.  Do not freeze Vacutainer® tubes.
• Red-top tube, plastic
This tube is a plastic Vacutainer containing a clot activator but no anticoagulants, preservatives, or separator material.  It is used for collection of serum for selected laboratory tests as indicated.  If the specimen requirement for a test is red-top tube(s), do not use  gold-top/SST® tube(s), as the gel separator may interfere with analysis.
• Red-top tube, glass
This tube is a plain glass Vacutainer® containing no clot activators, anticoagulants, preservatives or separator material.  These tubes can be used for Blood Bank tests.
• Pink-top tube (EDTA)
This tube contains EDTA as an anticoagulant. These tubes are preferred for blood bank tests.
NOTE:
After the tube has been filled with blood, immediately invert the tube 8-10 times to mix and ensure adequate anticoagulation of the specimen.
• Light green-top tube (lithium heparin)
This tube contains lithium heparin and gel separator used for the collection of heparinized plasma for routine chemistry tests.
NOTE:
After the tube has been filled with blood, immediately invert the tube 8-10 times to mix and ensure adequate anticoagulation of the specimen.
• Dark green-top tube (sodium heparin)
This tube contains sodium heparin used for the collection of heparinized plasma or whole blood for special tests.
NOTE:
After the tube has been filled with blood, immediately invert the tube 8-10 times to mix and ensure adequate anticoagulation of the specimen.
• Grey-top tube (potassium oxalate/sodium fluoride)
This tube contains potassium oxalate as an anticoagulant and sodium fluoride as a preservative – used to preserve glucose in whole blood and for some special chemistry tests.
NOTE:
After the tube has been filled with blood, immediately invert the tube 8-10 times to mix and ensure adequate anticoagulation of the specimen.
• Lavender-top tube (EDTA)
This tube contains EDTA as an anticoagulant - used for most hematological procedures. These tubes are preferred for molecular tests.
NOTE: After the tube has been filled with blood, immediately invert the tube 8-10 times to mix and ensure adequate anticoagulation of the specimen.To avoid RBC shrinkage due to excess EDTA (with resulting changes in HCT and RBC indices values) and possible dilutional effect, the tubes should be filled with the proper amount of blood for the size of tube used. Tubes with various draw volumes are available (2.0 mL, 3.0 mL, 5.0 mL and 0.75 mL microvettes) to assure proper ratio of EDTA to blood, it is recommended that the tubes contain no less than one-half of the stated volume.
• Royal blue-top tube
There are two types of royal blue-top Monoject® tubes – one with the anticoagulant EDTA and the other plain.  These are used in the collection of whole blood or serum for trace element analysis. Refer to the individual metals in the individual test listing to determine the tube type necessary.
• Yellow-top tube (ACD)
This tube contains ACD, which is used for the collection of whole blood for special tests.
NOTE:
After the tube has been filled with blood, immediately invert the tube 8-10 times to mix and ensure adequate anticoagulation of the specimen.
• Pearl white-top tube plasma preparation tube (PPT)
This tube contains EDTA and a special polyester material - used for the collection of plasma for molecular (PCR) tests.
NOTE:
After the tube has been filled with blood, immediately invert the tube 8-10 times to mix and ensure adequate anticoagulation of the specimen.

Special Collection Tubes: Some tests require specific tubes for proper analysis. Please contact the laboratory prior to patient draw to obtain the correct tubes for metal analysis or other tests as identified in the individual test listings.

Microtainer ®  tubes (pediatric bullet tubes)*

 Microtainer ® tube Color Gold Red Light Green (amber or clear) Lavender Light Green (without gel barrier) Additive No additive w/ gel barrier No additive Lithium Heparin w/ gel barrier K2EDTA Lithium Heparin without gel barrier Volume 0.5 mL 0.5 mL 0.5 mL 0.5 mL .04 mL

*Microtainer is a registered trademark of Becton, Dickinson and Company

Microbiological Collection Containers

Anaerobic Transport Media: Tube with soft agar and reducing agents designed to maintain viability of anaerobic organisms. Fluid can be injected through the diaphragm cap into the tube. To transport swab specimens or tissue, remove the cap, place the specimen into the tube (break off the swab stem if needed) and replace and tighten cap.

Blood Culture Media: Draw 20 mL of blood and aseptically inoculate 10 mL into each of the 2 bottles: BACTEC PLUS (blue label) and BACTEC LYTIC (purple label). Use an alcohol prep to clean the top of each bottle before and after inoculation.

Charcoal Amies Medium With Swab: This swab is used for the collection and transport of specimens for Bordetella isolation. The specimen of choice is secretions collected from the posterior nasopharynx. Culture Swab™ must be submitted for direct examination.

Chlamydia Culture Transport Medium: Use the swab provided to collect the specimen and inoculate the transport medium. If specimen transport is delayed, refrigerate after inoculation.

Chlamydia trachomatis, MicroTrak ®  Direct Stain Specimen Collection Kit: This is used in the collection of specimens for analysis by the MicroTrak C trachomatis direct specimen test. The kit contains slide, swabs, cytology brush, and fixative. The directions for use are on the package.

HSV 1, HSV 2, VZV MicroTrak Direct Stain Specimen Collection Kit: For use in the collection of specimens for analysis by the HSV1/HSV2 and VZV direct specimen typing test. This test is for external lesions only. The kit contains slides, swabs, fixative and directions for use. Collection kits are available in microbiology.

Isolator Microbial Tube (adult and pediatric): These tubes are used for the collection of blood specimens for the isolation of fungi and mycobacteria. Transport to the laboratory at ambient temperature.

Viral Culturette ® : This sterile swab is used for the collection and transport of herpes and viral specimens. Transport to the laboratory on wet ice.

Urine Collection

Random Collection: For routine and microscopic evaluation, a clean catch or midstream specimen is preferred.

Inpatients: The patient should void a small amount of urine, which is discarded. Collect urine in a clean container before voiding is completed. The container should be capped, labeled and refrigerated.

Outpatients and Referral Patients: After collection, the transfer of urine into preservative tubes should happen at the collection site. Mix the urine and peel back the protective sticker on the blue cap to expose the cannula. Fill the tubes in this order: gray for culture & sensitivity, yellow-red marble top for urinalysis and non-additive red top . The tube is filled by pushing the tube stopper side down onto the exposed cannula. Each tube has a line indicating the “minimum draw.”

Urine Collection for Chlamydia/Gonorrhea PCR:  Patient must not have voided for at least 2 hours.  The first stream of urine is collected and submitted for testing.

24-hour Urine Collection: UCI Pathology Services provides 24-hour urine collection containers.

Use the following procedure for the correct specimen collection and preparation:

1. Warn the patient of the presence of potentially hazardous preservatives in the collection container. Instruct the patient to discard the first morning specimen and to record the time of voiding.
2. The patient should collect all subsequent voided urine for the remainder of the day and night.  The first-morning specimen on day two should be collected at the same time as noted on day one.
3. Mix well before aliquoting and provide the total volume of the 24-hour urine collection or send the complete 24-hour collection to the laboratory.

Please note that specimens for Cytology should be submitted directly to the Department of Anatomic Pathology (not the UCSF Clinical Labs) with a paper copy of the APeX requisition.

CSF should be collected and transported to the laboratory in the special vials provided in the lumbar puncture kit. If unavailable, a plastic vial with a black screw top cap is acceptable. Each vial should be labeled with patient information (full name and medical record number are minimum requirements) and also list the name of the person who collected the specimen and the date it was collected. Note: CSF samples for Beta Amyloid 42/Tau Protein Analysis must be collected in a special polypropylene tube to prevent adherence of the protein to the sides of the tube. The tubes are stocked in the Memory & Aging clinic and are available from laboratory processing areas at each hospital.

Unless otherwise specified, requested tests will be done on selected tubes as follows:

• Tube #1 Chemistry & Immunology tests
• Tube #2 Microbiology cultures/tests
• Tube #3 Cell counts and differentials
• Tube #4 Cytology (Please submit directly to Anatomic Pathology with a paper copy of the requisition)

Cell counts are preferably performed on Tube #3 to reduce the impact of blood contamination secondary to the procedure itself. Counts on multiple tubes are rarely required unless Tube #3 is visibly bloody at which point a cell count on Tube #1 may be requested. A decrease in counts between Tube #1 and Tube #3 suggests a traumatic tap. In this circumstance cell counts should be interpreted with caution. Note: cell counts will not be performed on tube #1 if that sample is grossly bloodier than tube #3.

## Top 5 Anticoagulants Used in Hematology Laboratory | Biology

The following points highlight the top five anticoagulants that are commonly used in hematology. The anticoagulants are: 1. Double Oxalate 2. Ethylene Di-Amine Tetra Acetic Acid 3. Heparin 4. Sodium Citrate 5. Sodium Fluoride.

#### Anticoagulant # 1. Double Oxalate:

0.5 anticoagulant for 5 ml of blood.

This anticoagulant removes the free calcium ion from solution through the addition of ammonium and potassium oxalate. Calcium is precipitated as insoluble calcium oxalate.

i. 1-2 gram ammonium oxalate and 0.8gm potassium oxalate are dissolved in 100ml of distilled water.

ii. 0.5ml of this solution is added to each of a series of tubes and evaporated to dryness at 37°C higher temperature, decompose the exalted.

Blood taken into this anticoagulant is unsuitable for morphological examination the red cells commerce to crenate and the white exhibit bizarre nuclear patterns.

#### Anticoagulant # 2. Ethylene Di-Amine Tetra Acetic Acid (EDTA):

EDTA can be found in three salt forms:

Also, EDTA can be crystalline or liquid. Liquid EDTA tubes, requires specific filling volume to avoid dilution effect. So, blood: anticoagulant ratio must be maintained (this is applicable to all anticoagulants). EDTA is also known as Versene or Sequestrene.

EDTA acts by chelating/removing ionized calcium (calcium is required for blood to clot, so when it is removed blood will not clot). Generally tri-Potassium EDTA is better than di-Sodium EDTA and di-Lithium EDTA.

Always, be sure to mix blood with anticoagulant in a manner that guarantee proper complete mixing, by gentle repeated inversion of the tube, inversion for at least 20 times, do not shake or use vigorous inversion, since this may cause hemolysis, and disintegration of cells, and the final effect will be erroneous low results for cellular components of blood EDTA is the most commonly used anticoagulant in the hematology laboratory, and is the anticoagulant of choice for the CBC.

Excess EDTA (i.e. more EDTA, you fill less blood volume, so EDTA is in excess), causes shrinkage of RBC’s, causing falsely/erroneously reduced hematocrit (HCT), and subsequent increase in MCHC and decrease in MCV (MCV and MCHC are RBC indices that will be studied later).

Platelets are also affected, they will swell and subsequently disintegrate, causing erroneously high platelet count, since platelets will be disintegrated into more than one fragment, and each fragment will be counted as one platelet

#### Anticoagulant # 3. Heparin:

Heparin is an acid mucopolysaccharide, it acts by complexing with antithrombin to prevent blood clotting (antithrombin is one of the natural/physiological inhibitors of blood coagulation). It is not suitable for blood films staining, since it gives too blue coloration to the background, when films are stained with Romanovsky stains, also, heparin may cause leukocyte and platelet clumping, this is why heparin is not suitable for routine hematology tests.

It is the preferred anticoagulant for osmotic fragility test. Heparin also is used in capillary tubes for spun hematocrit (HCT) (heparin cover the entire capillary tube glass), these capillary tubes are also called microhematocrit capillary tubes. Heparin is also used for L.E. cell preparation (L.E. = Lupus Erythromatosus).

i. Heparin is found in basophil and mast cell granules

ii. Heparin is used therapeutically as an in vivo anticoagulant.

Anticoagulants Commonly Used in the Hematology Laboratory and their Use:

No. Anticoagulant Hematology Laboratory Use Universal Color Code

1. EDTA Routine Hematology Procedures. Lavender, Pink

2. Sodium citrate Coagulation, Platelets Tests, ESR. Blue

3. Heparin Osmotic Fragility, Spun Hematocrit Green, Brown

a. 0.4gm of prepared heparin is dissolved in 100ml of distilled water.

b. 0.25ml of this solution is added to each of a series tubes and evaporated to dryness of 37°C.

#### Anticoagulant # 4. Sodium Citrate:

This is most commonly used anticoagulant, mainly used to prepare citrated plasma for the evaluation of coagulation disorders as it is not inhibitory to calcium ions.

Is the anticoagulant of choice for coagulation and platelet function tests, also is used for ESR (erythrocyte sedimentation rate test). It acts by precipitating calcium, thus it will not be available for clotting process.

It came in a liquid form, as 3.8% tri-sodium citrate. For coagulation testing, the ratio of 9 volumes of blood to one volume of anticoagulant (9 volumes blood: 1 volume anticoagulant) is very critical (very important), as variation from this ratio may cause errors. For ESR (4) volumes of blood to one volume of anticoagulant is used (4: 1).

Generally, this anticoagulant is not suitable for routine hematology testing. From this we conclude that sodium citrate acts as anticoagulant and as diluent (as in the case of ESR). Because of its dilution effect it can’t be used for CBC.

3.8g Trisodium citrate is dissolved in 100ml of distilled water

#### Anticoagulant # 5. Sodium Fluoride:

This anti-coagulant is used for preparing blood specimens for the determination of glucose and urea in plasma by non-enzymatic methods. Fluroide inhibits glycolic enzymes and thereby prevents loss of glucose during transportation or delay in specimen handling. As fluoride is not a strong anticoagulant, it is mixed with oxalate.

## Which sample tube should be used for routine glucose determination?

Background: Glucose is one of the most frequently requested analytes in clinical laboratory. Blood glucose analysis is affected from in vitro glycolysis. In order to determine the most suitable blood collection tube for this purpose we have compared different tubes: sodium fluoride, lithium heparin, sodium fluoride/citrate buffer containing tubes and serum with clot activator tube for the measurement of glucose when the tube has been kept at room temperature (RT) for up to 4h.

Methods: Venous blood was collected from 49 healthy volunteers into Sarstedt S-Monovettes for glucose analysis. Reference plasma glucose was determined in a lithium heparin tube and immediately placed in an ice/water slurry. Within 10min it was centrifuged at 4°C and plasma was separated from the blood cells. Samples have been preserved at RT for 1, 2 and 4h after drawing. Glucose has been determined using a hexokinase method.

Results: Glucose levels tested in a serum with clot activator tube, in lithium heparin and in sodium fluoride/sodium EDTA tubes when compared with lithium-heparin reference plasma did not meet the desirable bias for glucose (±1.8%) when kept at RT for up to 4h. GlucoEXACT tubes, when corrected by the Sarsted recommended factor of 1.16, showed a mean (95% CI) bias of +0.96% (0.45-1.47) at 1h, +1.40% (0.88-1.93) at 2h and +0.95% (0.44-1.46) at 4h, reaching the analytical goal for the desirable bias.

Conclusions: Samples collected into GlucoEXACT tubes containing sodium fluoride/citrate buffer liquid mixture are equivalent to those collected in reference plasma tubes avoiding glycolysis completely and within a 4h delay in plasma separation.

Keywords: Citrate acidification Glucose Glycolysis inhibition Hexokinase Preanalytical conditions Sodium fluoride Stability.

## 2.1. Background information on best practices in phlebotomy

Best practices in phlebotomy involve the following factors:

availability of appropriate supplies and protective equipment

avoidance of contaminated phlebotomy equipment

cooperation on the part of patients

This is the most important part of carrying out any procedure, and is usually done at the start of a phlebotomy session.

### 2.1.2. Using an appropriate location

The phlebotomist should work in a quiet, clean, well-lit area, whether working with outpatients or inpatients.

### 2.1.3. Quality control

Quality assurance is an essential part of best practice in infection prevention and control (1). In phlebotomy, it helps to minimize the chance of a mishap. Table 2.1 lists the main components of quality assurance, and explains why they are important.

#### Table 2.1

Elements of quality assurance in phlebotomy.

### 2.1.4. Quality care for patients and health workers

Several factors can improve safety standards and quality of care for both patients and health workers, and laboratory tests. These factors, discussed below, include:

#### Availability of appropriate supplies and protective equipment

Procurement of supplies is the direct responsibility of the administrative (management) structures responsible for setting up phlebotomy services. Management should:

Several safety-engineered devices are available on the market such devices reduce exposure to blood and injuries. However, the use of such devices should be accompanied by other infection prevention and control practices, and training in their use. Not all safety devices are applicable to phlebotomy. Before selecting a safety-engineered device, users should thoroughly investigate available devices to determine their appropriate use, compatibility with existing phlebotomy practices, and efficacy in protecting staff and patients (12, 33). Annex B provides further information on infection prevention and control, safety equipment and best practice Annex C provides a comprehensive guide to devices available for drawing blood, including safety-engineered equipment.

For settings with low resources, cost is a driving factor in procurement of safety-engineered devices.

Where safety-engineered devices are not available, skilled use of a needle and syringe is acceptable.

#### Availability of post-exposure prophylaxis

Accidental exposure and specific information about an incident should be recorded in a register.

Support services should be promoted for those who undergo accidental exposure. PEP can help to avert HIV and hepatitis B infections (13, 27). Hepatitis B immunization should be provided to all health workers (including cleaners and waste handlers), either upon entry into health-care services or as part of PEP (34). Annex D has details of PEP for hepatitis B and HIV.

#### Avoidance of contaminated phlebotomy equipment

Tourniquets are a potential source of methicillin-resistant Staphylococcus aureus (MRSA), with up to 25% of tourniquets contaminated through lack of hand hygiene on the part of the phlebotomist or reuse of contaminated tourniquets (35). In addition, reusable finger-prick devices and related point-of-care testing devices (e.g. glucometers) contaminated with blood have been implicated in outbreaks of hepatitis B (4, 5, 36).

To avoid contamination, any common-use items, such as glucometers, should be visibly clean before use on a patient, and single-use items should not be reused.

#### Training in phlebotomy

All staff should be trained in phlebotomy, to prevent unnecessary risk of exposure to blood and to reduce adverse events for patients.

#### Patient cooperation

One of the essential markers of quality of care in phlebotomy is the involvement and cooperation of the patient this is mutually beneficial to both the health worker and the patient.

Clear information – either written or verbal – should be available to each patient who undergoes phlebotomy. Annex F provides sample text for explaining the blood-sampling procedure to a patient.

### 2.1.5. Quality of laboratory sampling

Factors that influence the outcome of laboratory results during collection and transportation include:

## Specimen collection and processing procedures

Proper specimen collection and handling is an integral part of obtaining a valid and timely laboratory test result. Specimens must be obtained in the proper tubes or containers, correctly labeled, and then promptly transported to the laboratory.

### Obtaining reliable and accurate laboratory test results

Physicians and others responsible for obtaining specimens and transporting them to the laboratory have a vital role in ensuring that laboratory test results are valid. The following are essential safeguards for your patients.

1. Avoid patient identification errors

Use at least two patient identifierswhen administering medications, blood, or blood components.

Label containers used for blood and other specimens in the presence of the patient.

–Joint Commission National Patient Safety Goal 1, Elements of Performance, 2015.

Identify the patient prior to collecting a sample. Check armbands. Acceptable identifiers may include the patient’s full name, date of birth, or medical record number.

2. Draw the tubes in the proper sequence

When multiple tubes are to be drawn from a single venipuncture using an evacuated tube system (e.g. BD Vacutainer® or Greiner Vacuette®), there is a correct sequence for blood collection that prevents cross-contamination of tube additives that could cause erroneous test results. The following should be used for both plastic and glass blood collection tubes.

1. Blood culture
2. Coagulation tube (blue top) (1)
3. Serum tube with or without clot activator, with or without gel (red or gold top)
4. Heparin tube with or without gel plasma separator (green top)
5. EDTA (purple top, pink top)
6. Oxalate and fluoride (gray top)
7. Other special tubes

(1) All blue tops collected for coagulation assays which do not have a blood culture collected first must have a discard tube collected prior to filling. Discard tube should be another blue top in which greater than 1 mL of blood is collected.

All tubes must be gently inverted 10 times end-to-end immediately after collection.

3. Use proper containers for collection

Certain analyses require containers with preservatives and/or anticoagulants, while others do not. Using the wrong container often leads to erroneous results. See the test catalog for exact requirements.

4. Mix all tubes ten times by gentle inversion immediately after collection

5. Do not decant specimens from one type of container into another

Specimens must be submitted to the laboratory in the container used originally for collection.

6. Deliver specimens to the laboratory promptly

Valid measurement of analytes in serum or plasma requires prompt separation from the blood cells. When left unseparated, analytes shift between the cells and the plasma or serum and glucose is consumed. Some analytes are unstable at room temperature. Drawing extra tubes of blood on patients and holding them as a contingency against some unforeseen need for more tests can lead to erroneous results and is a dangerous practice that should be avoided.

### For specimens drawn off campus

Red and gold top tubes must stand for 30 minutes to allow for complete clotting. They must then be centrifuged and the serum separated and refrigerated until delivered to the laboratory. Check specific test information in the manual to determine if serum should be frozen.

Purple top tubes for CBCs may be kept at room temperature for up to 8 hours. After 8 hours, refrigerate until delivery. Stable 36 hours refrigerated. For tests drawn in purple top tubes other than CBCs, please check specific test stability.

Green top tube handling depends on the specific test ordered. Check specific test directions.

### Laboratory specimen storage temperature requirements

 Storage Method Centigrade (Celsius) Temperature Range Refrigerated 2 – 8 º C Frozen less than or equal to -20 º C Room/ Ambient 20 – 25 º C

7. Avoid hemolysis

Erythrocytes contain certain analytes (LD, AST, K, ALT) in concentration many times higher than in plasma. When red cells are hemolyzed, there is a release of these analytes and dilution of plasma, resulting in erroneous laboratory values. Also, hemolysis may interfere in analytical methodologies.

8. Drawing samples from a line

If the sample is to be drawn from a line, be sure to draw approximately 5-10 mL for adults in a first “flush” syringe (20 mL to clear any heparin from the line if coagulation tests are desired). Then draw the syringe for the desired tests. The “flush” may be given back to the patient if needed.

## How to Send a Specimen

When submitting specimens to the University of Washington Department of Laboratory Medicine, please complete a requisition order form and send with the specimen.

NOTE: Each patient's specimen and corresponding requisition form must be contained in a single, separate specimen bag. We cannot accept specimens and paperwork from multiple patients if they are gathered together in a single bag.

For information, assistance, or for pre-addressed labels, call (206) 520-4600 or (800) 713-5198.

### Labeling Specimens

• Please affix patient label vertically with the last name closer to the top end of the tube.
• Label or tag each specimen in English with at least two of the following: patient name, date of birth and/or unique I.D. number corresponding to the request form.
• Use dark, indelible ink. Be sure label or tag is secure.
• Use a separate request form for each specimen type or clearly mark specimen types on form.

All specimens must be properly labeled to ensure patient safety and prevent errors in patient diagnosis and treatment based on misidentified labels. Mislabeled/unlabeled specimens will be rejected.

#### Unacceptable Specimens Include:

• Specimens that are not labeled.
• Specimens labeled with a patient name and/or patient identification number different from that on the accompanying laboratory request form.
• Specimens drawn from correct patient but labeled with wrong name and identification number.
• Specimens that have labels and requisitions that agree but have been drawn from the wrong patient.
• Specimens or requisitions with <2 matching patient identifiers.
• Two or more specimens sent in the same specimen bag.

Whenever an unacceptable specimen is received, the ordering location will be notified of the error and requested to send a new specimen. Re-labeling will not be allowed except in rare instances where recollection may cause undue risk to the patient (e.g., CSF from lumbar puncture, tissues from a biopsy, or other similarly obtained irreplaceable samples). For these rare cases a clinical pathologist or designee will have to approve the request.

### Shipping Specimens

To avoid specimen rejection because of improper handling and degradation in transit, please follow the specimen requirement instructions in the test menu section of this guide. Any questions should be directed to Client Support Services (206) 520-4600 or (800) 713-5198. All specimens must be bagged for handling. The request forms must be in a separate compartment or bag.

NOTE: Each patient's specimen and corresponding requisition form must be contained in a single, separate specimen bag. We cannot accept specimens and paperwork from multiple patients if they are gathered together in a single bag.

#### Clinical Specimens & Biological Substances

Any human or animal material, including but not limited to, excreta, secreta, blood and its components, tissue and tissue fluids, being shipped for diagnostic or investigational purposes must be packaged and shipped correctly. To determine the proper packaging, see Transport and Packaging of Specimens for details. All specimens prepared for transport to the University of Washington Medical Center or Harborview Medical Center should follow these general instructions. Please follow the appropriate directions whether you intend to use our courier, the bus, airfreight, or mail services (clients should follow any additional guidelines established by the U.S. Postal Service or commercial carrier they are using for safe transport of biological specimens). All specimens should be considered biohazards and handled accordingly.

NOTE: OSHA requires that all shipments containing clinical specimens be marked with a "Biohazard" label. Labeled bags for shipments sent to Client Support Services will be provided on request. Containers for shipping are also available through Client Support Services (206) 520-4600 or (800) 713-5198, upon request.

#### Packaging and Transport

As of March 1, 2003 we would like all clients sending specimens to us to adopt a uniform specimen packaging procedure. In order to insure the safety of our couriers, and to make our processing workflow as streamlined and trouble-free as possible, we ask that you follow these guidelines:

• All specimens should be contained in sealed plastic specimen bags.
• The specimen bags should display the Biohazard logo.
• Each specimen bag should contain enough absorbent material to contain all of the contents of specimen tubes in case of breakage.
• Each specimen bag should contain only ONE patient's specimen.
• Each specimen should be accompanied by the corresponding requisition sheet, folded and placed in the outside pocket of the specimen bag.

#### Frozen Specimens

Certain specimens must be shipped frozen to maintain stability of the analyte. Shipping containers should be filled with dry ice to prevent samples from thawing in case of shipping delays. Specimens should be shipped in containers that have been tested, safe for use with dry ice, and are known to keep samples frozen for at least 48 hours. Do not ship frozen specimens in glass containers unless specified under specimen requirements. Mark the outside of the container with label: FROZEN MATERIAL - DO NOT THAW.

#### Refrigerated Specimens

For refrigerated specimens, use the same Styrofoam mailing container as is provided for frozen specimens. Place on a coolant pack and fill the vacant space with any type of packaging material. We request that coolant packs be shipped in plastic bags. Mark the outside of the container: REFRIGERATED MATERIAL - DO NOT FREEZE.

#### Ambient Temperature Specimens

These specimens do not require special temperature conditions, but should not be allowed to freeze. However, safe specimen transport guidelines must be followed when specimens are submitted.

Specimens which contain radioisotopes will be accepted for testing only when prior arrangements have been made and the specimens are clearly labeled so as to identify the contaminating isotope, the dose and compound administered to the patient, and the date of administration. To minimize hazards, specimens which are very "hot", e.g. those taken immediately after ablative therapy, may be held until they are less radioactive or they may be analyzed by methods which are not routine in our laboratories. Such options will be discussed with you when you make arrangements to send the specimen.

#### Courier Services

Courier service is provided at no charge* in the areas near Auburn, Bellevue, Bellingham, Bothell, Edmonds, Everett, Kent, Kirkland, Mt. Vernon, Olympia, Renton, Seattle, and Tacoma. The courier route includes bus and air freight depots. Stops are made at Sea-Tac Airport as needed. To schedule a pickup, please telephone us. Stops are made at the Greyhound Bus Terminal in the morning and afternoon, Monday through Friday. See Transport and Packaging of Specimens.

There is no STAT courier service.

All fees incurred for specimen transport other than via our courier service are the responsibility of the patient/sending location.

*Please contact Client Support Services office at 206.520.4600 or 1 800 713-5198 if you intend to use our courier service for non-departmental delivery.

### Shipping Addresses for Outside Specimens

The address you select for packages will vary according to the urgency of the package and the time it is sent. Routine specimens for ANY departmental laboratory (including Virology, Toxicology, Immunology, Microbiology, Hematopathology etc.) may be sent to the address below:

• UW Medicine
• Department of Laboratory Medicine
• 1959 NE Pacific Street, room NW220
• Seattle, WA 98195
• Tel: (206) 520-4600 or 1 (800) 713-5198

For packages sent by BUS or PLANE, and needing to be picked up, please add:

It is recommended that you notify the Client Support Services Office (206) 520-4600 or (800) 713-5198 during business hours, 8 AM-5 PM Monday through Friday, prior to sending packages by bus or plane. At times outside of these hours, please call the laboratory directly at (206) 598-6224.

#### Toxicology Stats

Because some of our laboratories are in separate locations, STAT toxicology specimens may be shipped directly to Harborview Medical Center. Please call (206) 744-3451 before shipping STAT specimens.

• Harborview Medical Center
• Clinical Laboratory, Room GWH47
• 325 9th Avenue
• Seattle, WA 98104
• Call on Arrival - 206.731-3451 RUSH

When sending to this address, please ensure all of the following is done:

1. Do not send routine specimens to this address.
2. Mark STAT or RUSH on the outside of the package as well as on the request form.
3. Include the telephone number on the outside of the package.

## Procedure of Indirect Coombs Test

1. Label three test tubes as T (test serum) PC (Positive control) and NC (negative control).
2. In the tube labeled as T (Test), take 2 drops of test serum.
3. In the test tube labeled as PC (Positive control), take 1 drop of anti D serum.
4. In the test tube labeled as NC (Negative control), take 1 drop of normal saline.
5. Add one drop of 5 % saline suspension of the pooled ‘O’ Rho (D) positive cells in each tube.
6. Incubate all the three tubes for one hour at 37°C.
7. Wash the cells three times in normal saline to remove excess serum with no free antibodies, (in the case of inadequate washings of the red cells, negative results may be obtained).
8. Add two drops of Coombs serum (anti human serum) to each tube.
9. Keep for 5 minutes and then centrifuge at 1,500 RPM for one minute.
10. Resuspend the cells and examine macroscopically as well as microscopically.

### Result Interpretation of Coombs Test

Negative Result:

No clumping of cells (no agglutination). This means you have no antibodies to red blood cells.

Positive Result:

Clumping (agglutination) of the blood cells during a direct Coombs test means that you have antibodies on the red blood cells and that you may have a condition that causes the destruction of red blood cells by your immune system (hemolysis). This may be due to

• Hemolytic anemia,
• Chronic lymphocytic leukemia or similar disorder,
• Erythroblastosis fetalis (hemolytic disease of the newborn),
• Infectious mononucleosis,
• Mycoplasmal infection,
• Syphilis,
• Systemic lupus erythematosus and
• Transfusion reaction, such as one due to improperly matched units of blood.

## Lab 12: Isolation and Identification of Enterobacteriaceae and Pseudomonas, Part 1

Labs 12 and 13 deal with opportunistic and pathogenic fermentative Gram-negative bacilli that are members of the bacterial family Enterobactereaceae, as well as nonfermentative Gram-negative bacilli such as Pseudomonas and Acinetobacter.

A. ENTEROBACTERIACEAE: THE FERMENTATIVE, GRAM-NEGATIVE, ENTERIC BACILLI

Bacteria belonging to the family Enterobacteriaceae are the most commonly encountered organisms isolated from clinical specimens. The Enterobacteriaceae is a large diverse family of bacteria belonging to the order Enterobacteriales in the class Gammaproteobacter of the phylum Proteobacter. Medically important members of this family are commonly referred to as fermentative, Gram-negative, enteric bacilli, because they are Gram-negative rods that can ferment sugars. Many are normal flora of the intestinal tract of humans and animals while others infect the intestinal tract. Members of this family have the following characteristics in common:

1. They are Gram-negative rods (see Fig. 1)
2. If motile, they possess a peritrichous arrangement of flagella (see Fig. 2)
3. They are facultative anaerobes
4. With few exception, they are oxidase negative
5. All species ferment the sugar glucose but otherwise vary widely in their biochemical characteristics
6. Most reduce nitrates to nitrites.

For further information on the Gram-negative cell wall, see the following Learning Object in your Lecture Guide:

At least forty-four genera and over 130 species of Enterobacteriaceae have been recognized. Some of the more common clinically important genera of the family Enterobacteriaceae include:

 Salmonella Citrobacter Morganella Shigella Enterobacter Yersinia Proteus Serratia Edwardsiella Escherichia Klebsiella Providencia

Several genera of Enterobacteriaceae are associated with gastroenteritis and food-borne disease. These include:

• Salmonella,
• Shigella,
• certain strains of Escherichia coli, and
• certain species of Yersinia.

All intestinal tract infections are transmitted by the fecal-oral route.

There are two species of Salmonella, Salmonella enterica and Salmonella bongori. Any infection caused by Salmonella is called a salmonellosis. Non-typhoidal Salmonella accounts for an estimated 520 cases per 100,000 population (approximately 1,600,000 cases) per year in the U.S. and at least 500 die. Since many different animals carry Salmonella in their intestinal tract, people usually become infected from ingesting improperly refrigerated, uncooked or undercooked poultry, eggs, meat, dairy products, vegetables, or fruit contaminated with animal feces.

E nteritis is the most common form of salmonellosis. Symptoms generally appear 6-48 hours after ingestion of the bacteria and include vomiting, nausea, non-bloody diarrhea, fever, abdominal cramps, myalgias, and headache. Symptoms generally last from 2 days to 1 week followed by spontaneous recovery. All species of Salmonella can cause bacteremia but S. enterica serotype Typhi, isolated only from humans, frequently disseminates into the blood causing a severe form of salmonellosis called typhoid fever. About 400 cases of typhoid fever occur each year in the U.S. but approximately 75% of these are acquired while traveling internationally.

Salmonella serotyping is a subtyping method of identification based on the identification of distinct cell wall, flagellar, and capsular antigens with known antiserum, as will be discussed in Lab 17. Salmonella serotypes Enteritidis and Typhimurium are the two most common serotypes in the United States, accounting for approximately 35 to 40% of all infections confirmed by laboratory culture. As mentioned above, S. enterica serotype Typhi is responsible for typhoid fever.

Any Shigella infection is called a shigellosis. Unlike Salmonella, which can infect many different animals, Shigella only infects humans and other higher primates. There are approximately 14,000 laboratory cases of shigellosis a year reported in the US with an estimated 450,000 total cases and 70 deaths.

Shigellosis frequently starts with a watery diarrhea, fever, and abdominal cramps but may progress to dysentery with scant stools containing blood, pus, and mucus. The incubation period is 1-3 days. Initial profuse watery diarrhea typically appears first as a result of enterotoxin. Within 1-2 days this progresses to abdominal cramps, with or without bloody stool. Classic shigellosis presents itself as lower abdominal cramps and stool abundant with blood and pus develops as the Shigella invade the mucosa of the colon.

Escherichia coli is one of the dominant normal flora in the intestinal tract of humans and animals. Some strains, however, can cause infections of the intestines while others are capable of causing infections outside the intestines. Extraintestinal pathogenic E. coli cause such opportunistic infections as urinary tract infections, wound infections, and septicemia and will be discussed in greater detail below. Intestinal or diarrheagenic E. coli cause infections of the intestinal tract. Diarrheagenic E. coli include:

• Enterotoxigenc E. coli (ETEC) produce enterotoxins that cause the loss of sodium ions and water from the small intestines resulting in a watery diarrhea. It is an important cause of diarrhea in impoverished countries. Over half of all travelers' diarrhea is due to ETEC almost 80,000 cases a year in the U.S.
• Enteropathogenic E. coli (EPEC) causes an endemic diarrhea in in impoverished countries, especially in infants younger than 6 months of age. The bacterium disrupts the normal microvilli on the epithelial cells of the small intestines resulting in maladsorbtion and diarrhea. They do not produce enterotoxin or shiga toxin and are not invasive. It is rare in industrialized countries.
• Enteroaggregative E. coli (EAEC) is a cause of endemic diarrhea in children in impoverished countries and industrialized countries. It is also responsible for a persistant diarrhea in people infected with HIV. It probably causes diarrhea by adhering to mucosal epithelial cells of the small intestines and interfering with their function.
• Enteroinvasive E. coli (EIEC) invade and kill epithelial cells of the colon usually causing a watery diarrhea but sometimes progressing to a dysentery-type syndrome with blood in the stool. It occurs mostly in impoverished countries and is rare in industrialized countries.
• Enterohemorrhagic E. coli (EHEC), such as E. coli 0157:H7, produce a shiga toxin that kills epithelial cells of the colon causing hemorrhagic colitis, a bloody diarrhea. In rare cases, the shiga toxin enters the blood and is carried to the kidneys where, usually in children, it damages vascular cells and causes hemolytic uremic syndrome. E. coli 0157:H7 is thought to cause more than 20,000 infections and up to 250 deaths per year in the U.S.

Several species of Yersinia, such as Y. enterocolitica and Y. pseudotuberculosis are also causes of diarrheal disease.

Many other genera of the family Enterobacteriaceae are normal microbiota of the intestinal tract and are considered opportunistic pathogens. The most common genera of Enterobacteriaceae causing opportunistic infections in humans are:

• Escherichia coli,
• Proteus,
• Enterobacter,
• Klebsiella,
• Citrobacter, and
• Serratia.

They act as opportunistic pathogens when they are introduced into body locations where they are not normally found, especially if the host is debilitated or immunosuppressed. They all cause the same types of opportunistic infections, namely:

• urinary tract infections,
• wound infections,
• pneumonia, and
• septicemia.

These normal flora Gram-negative bacilli, along with Gram-positive bacteria such as Enterococcus species (see Lab 14) and Staphylococcus species (see Lab 15), are among the most common causes of healthcare-associated infections (formerly called nosocomial infections).

According to the Centers for Disease Control and Prevention (CDC) Healthcare-associated infection's website, "In American hospitals alone, healthcare-associated infections account for an estimated 1.7 million infections and 99,000 associated deaths each year. Of these infections:

• 32 percent of all healthcare-associated infection are urinary tract infections (UTIs)
• 22 percent are surgical site infections
• 15 percent are pneumonia (lung infections)
• 14 percent are bloodstream infections"

Most patients who have healthcare-associated infections are predisposed to infection because of invasive supportive measures such as urinary catheters, intravascular lines, and endotracheal intubation.

By far, the most common Gram-negative bacterium causing nosocomial infections is Escherichia coli. E. coli causes between 70 and 90% of both upper and lower urinary tract infections (UTIs). It is also a frequent cause of abdominal wound infections and septicemia. Depending on the facility, E. coli is responsible for between 12% and 50% of all healthcare-associated infections.

However, according to a 2008 study, Enterobacteriaceae other than E. coli were responsible for 7 of the 10 most common Gram-negative organisms isolated from urinary tract, respiratory tract, and bloodstream infections from intensive care unit patients between 2002 and 2008 in the United States. These include Klebsiella pneumoniae (15%), Enterobacter cloacae (9%), Serratia marcescens (6%), Enterobacter aerogenes (4%), Proteus mirabilis (4%), Klebsiella oxytoca (3%), and Citrobacter freundii (2%). Furthermore, the National Healthcare Safety Network reported K. pneumoniae (6%) , Enterobacter spp. (5%), and K. oxytoca (2%) among the top 10 most frequently isolated health care-associated infections between the years between 2006 and 2007.

1. Urinary Tract Infections

The most common infection caused by opportunistic Enterobacteriaceae is a urinary tract infection (UTI). UTIs account for more than 8, 000,000 physician office visits per year in the U.S and as many as 100,000 hospitalizations. Among the nonhospitalized and nondebilitated population, UTIs are more common in females because of their shorter urethra and the closer proximity between their anus and the urethral opening. (Over 20 percent of women have recurrent UTIs.) However, anyone can become susceptible to urinary infections in the presence of predisposing factors that cause functional and structural abnormalities of the urinary tract. These abnormalities increase the volume of residual urine and interfere with the normal clearance of bacteria by urination. Such factors include prostate enlargement, sagging uterus, expansion of the uterus during pregnancy, paraplegia, spina bifida, scar tissue formation, and catheterization. Between 35 and 40 percent of all nosocomial infections, about 900,000 per year in the U.S., are UTIs and are usually associated with catheterization.

E. coli and Staphylococcus saprophyticus (a Gram-positive staphylococcus that will be discussed in Lab 15) cause around 90 percent of all uncomplicated UTIs. Most of the remaining uncomplicated UTIs are caused by other Gram-negative enterics such as Proteus mirabilis and Klebsiella pneumoniae or by Enterococcus faecalis (a Gram-positive streptococcus that will be discussed in Lab 14). E. coli is responsible for more than 50 percent of healthcare-associated UTIs. Other causes of hospital-acquired UTIs include other species of Enterobacteriaceae (such as Proteus, Enterobacter, and Klebsiella), Pseudomonas aeruginosa (discussed below), Enterococcus species (discussed in lab 14) , Staphylococcus saprophyticus (discussed in Lab 15), and the yeast Candida (discussed in lab 9).

The traditional laboratory culture standard for a UTI has been the presence ofmore than 100,000 CFUs (colony-forming units see Lab 4) per milliliter (ml) of midstream urine, or any CFUs from a catheter-obtained urine sample. More recently, this has been modified and counts of as few as 1000 colonies of a single type per ml or as little as 100 coliforms per ml are now considered as indicating a UTI.

2. Wound Infections

Wound infections are due to fecal contamination of external wounds or a result of wounds that cause trauma to the intestinal tract, such as surgical wounds, gunshot wounds, and knife wounds. In the latter case, fecal bacteria get out of the intestinal tract and into the peritoneal cavity causing peritonitis and formation of abcesses on the organs found in the peritoneal cavity.

3. Pneumonia

Although they sometimes cause pneumonia, the Enterobacteriaceae account for less than 5% of the bacterial pneumonias requiring hospitalization.

4. Bloodstream Infections

Gram-negative septicemia is a result of these opportunistic Gram-negative bacteria getting into the blood. They are usually introduced into the blood from some other infection site, such as an infected kidney, wound, or lung. Looking at patients that develop septic shock:

• Lower respiratory tract infections are the source in about 25% of patients.
• Urinary tract infections are the source in about 25% of patients.
• Soft tissue infections are the source in about 15% of patients.
• Gastrointestinal infections are the source in about 15% of patients.
• Reproductive tract infections are the source in about 10% of patients.
• Foreign bodies (intravascular lines, implanted surgical devices, etc.) are the source in about 5% of patients.

There are approximately 750,000 cases of septicemia per year in the U.S. and 200,000 cases of septic shock. Septic shock results in approximately 100,000 deaths per year in the U.S. Approximately 45 percent of the cases of septicemia are due to Gram-negative bacteria. Klebsiella, Proteus, Enterobacter, Serratia, and E. coli, are all common Enterobacteriaceae causing septicemia. (Another 45 percent are a result of Gram-positive bacteria (see Labs 14 and 15) and 10 percent are due to fungi, mainly the yeast Candida (see Lab 9).

In the outer membrane of the Gram-negative cell wall, the lipid A moiety of the lipopolysaccharide (LPS) functions as an endotoxin (see Fig 4 ). Endotoxin indirectly harms the body when massive amounts are released during severe Gram-negative infections. This, in turn, causes an excessive cytokine response.

1. TheLPS released from the outer membrane of the Gram-negative cell wall first binds to a LPS-binding protein circulating in the blood and this complex, in turn, binds to a receptor molecule (CD 14 ) found on the surface of body defense cells called macrophages (see Fig. 5) located in most tissues and organs of the body.

2. This is thought to promote the ability of the toll-like receptor TLR-4 to respond to the LPS, triggering the macrophages to release various defense regulatory chemicals called cytokines, including tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1), interleukin-6 (IL-6), and interleukin-8 (IL-8), and platelet-activating factor (PAF). The cytokines then bind to cytokine receptors on target cells and initiate inflammation and activate both the complement pathways and the coagulation pathway (see Fig. 5).

3. The complex of LPS and LPS binding protein can also attach to molecules called CD14 on the surfaces of phagocytic white blood cells called neutrophils causing them to release proteases and toxic oxygen radicals for extracellular killing. Chemokines such as interleukin-8 (IL-8) also stimulate extracellular killing. In addition, LPS and cytokines stimulate the synthesis of a vasodilator called nitric oxide.

D uring minor local infections with few bacteria present, low levels of LPS are released leading to moderate cytokine production by the monocytes and macrophages and in general, promoting body defense by stimulating inflammation and moderate fever, breaking down energy reserves to supply energy for defense, activating the complement pathway and the coagulation pathway, and generally stimulating immune responses (see Fig. 5). Also as a result of these cytokines, circulating phagocytic white blood cells such as neutrophils and monocytes stick to the walls of capillaries, squeeze out and enter the tissue, a process termed diapedesis. The phagocytic white blood cells such as neutrophils then kill the invading microbes with their proteases and toxic oxygen radicals.

However, during severe systemic infections with large numbers of bacteria present, high levels of LPS are releasedresulting inexcessive cytokine productionby the monocytes and macrophages and this canharm the body (see Fig. 6). In addition, neutrophils start releasing their proteases and toxic oxygen radicals that kill not only the bacteria, but the surrounding tissue as well. Harmful effects include high fever, hypotension, tissue destruction, wasting, acute respiratory distress syndrome (ARDS), disseminated intravascular coagulation (DIC), and damage to the vascular endothelium resulting in shock, multiple system organ failure (MOSF), and often death.

This excessive inflammatory response is referred to as Systemic Inflammatory Response Syndrome or SIRS. Death is a result of what is called the shock cascade. The sequence of events is as follows:

• Neutrophil-induced damage to the capillaries, as well as prolonged vasodilation, results in blood and plasma leaving the bloodstream and entering the surrounding tissue. This can lead to a decreased volume of circulating blood (hypovolemia).
• Prolonged vasodilation also leads to decreased vascular resistance within blood vessels while high levels of TNF inhibit vascular smooth muscle tone and myocardial contractility. This results in a marked hypotension.
• Activation of the blood coagulation pathway can cause clots called microthrombi to form within the blood vessels throughout the body causing disseminated intravascular coagulation (DIC).
• Increased capillary permeability as a result of vasodilation in the lungs, as well as neutrophil-induced injury to capillaries in the alveoli, lead to acute inflammation, pulmonary edema, and loss of gas exchange in the lungs (acute respiratory distress syndrome or ARDS). As a result, the blood does not become oxygenated.
• Hypotension, hypovolemia, ARDS, and DIC result in marked hypoperfusion.
• Hypoperfusion in the liver can result in a drop in blood glucose level from liver dysfunction.
• Hypoperfusion leads to acidosis and the wrong pH for enzymes involved in cellular metabolism resulting in cell death.
• Hypoperfusion also can lead to cardiac failure .

Collectively, this can result in :

• end-organ ischemia: a restriction in blood supply that results in damage or dysfunction of tissues or organs,
• multiple system organ failure (MSOF),
• death.

Both pili and surface proteins in the Gram-negative cell wall function as adhesins, allowing the bacterium to adhere intimately to host cells and other surfaces in order to colonize and resist flushing. Some Gram-negative bacteria also produce invasins, allowing some bacteria to invade host cells. Motility, capsules, biofilm formation, and exotoxins also play a role in the virulence of some Enterobacteriaceae.

For further information on virulence factors associated with various Enterobacteriaceae, see the following Learning Objects in your Lecture Guide:

Many of the Enterobacteriaceae also possess R (resistance) plasmids. These plasmids are small pieces of circular non-chromosomal DNA that may code for multiple antibiotic resistance In addition, the plasmid may code for a sex pilus, enabling the bacterium to pass R plasmids to other bacteria by conjugation. Between 50 and 60 percent of the bacteria causing healthcare-associated infections are antibiotic resistant.

For further information on bacterial resistance to antibiotics, see the following Learning Object in your Lecture E-Text:

The identification of lactose-fermenting Gram-negative rods belonging to the bacterial family Enterobacteriaceae (bacteria commonly referred to as coliforms) in water is often used to determine if water has been fecally contaminated and, therefore, may contain disease-causing pathogens transmitted by the fecal-oral route. The procedure for this is given in Appendix E.

B. PSEUDOMONAS AND OTHER NON-FERMENTATIVE GRAM-NEGATIVE BACILLI

Non-fermentative Gram-negative bacilli refer to Gram-negative rods or coccobacilli that cannot ferment sugars. The non-fermentative Gram-negative bacilli are often normal inhabitants of soil and water. They may cause human infections when they colonize immunosuppressed individuals or gain access to the body through trauma. However, less than one-fifth of the Gram-negative bacilli isolated from clinical specimens are non-fermentative bacilli. By far, the most common Gram-negative, non-fermentative rod that causes human infections is Pseudomonas aeruginosa. Pseudomonas belongs to the family Pseudomonadaceae in the order Pseudomonadales in the class Gammaproteobacter of the phylum Proteobacter.

Pseudomonas aeruginosa is also an opportunistic pathogen. It is a common cause of nosocomial infections and can be found growing in a large variety of environmental locations. In the hospital environment, for example, it has been isolated from drains, sinks, faucets, water from cut flowers, cleaning solutions, medicines, and even disinfectant soap solutions. It is especially dangerous to the debilitated or immunocompromised patient.

Like the opportunistic Enterobacteriaceae, Pseudomonas is a Gram-negative rod, it is frequently found in small amounts in the feces, and it causes similar opportunistic infections: urinary tract infections, wound infections, pneumonia, and septicemia. P aeruginosa is the fourth most commonly isolated nosocomial pathogen, accounting for 10% of all hospital-acquired infections. P. aeruginosa is responsible for 12 percent of hospital-acquired urinary tract infections, 16 percent of nosocomial pneumonia cases, and 10 percent of the cases of septicemia. In addition, P. aeruginosa is a significant cause of burn infections with a 60 percent mortality rate. It also colonizes and chronically infects the lungs of people with cystic fibrosis. Like other opportunistic Gram-negative bacilli, Pseudomonas aeruginosa also releases endotoxin and frequently possesses R-plasmids. A number of other species of Pseudomonas have also been found to cause human infections.

For further information on virulence factors associated with Pseudomonas, see the following Learning Objects in your Lecture Guide:

Other non-fermentative Gram-negative bacilli that are sometimes opportunistic pathogens in humans include Acinetobacter, Aeromonas, Alcaligenes, Eikenella, Flavobacterium, and Moraxella.

Acinetobacter has become a frequent cause of nosocomial wound infections, pneumonia, and septicemia. The bacterium has become well known as a cause of infections among veterans of the wars in Iraq and Afghanistan and is becoming a growing cause of nosocomial infections in the U.S. Acinetobacter is thought to have been contracted in field hospitals in Iraq and Afghanistan and subsequently carried to veteran's hospitals in the U.S. Because most species are multiple antibiotic resistant, it is often difficult to treat. Acinetobacter is commonly found in soil and water, as well as on the skin of healthy people, especially healthcare personnel. Although there are numerous species of Acinetobacter that can cause human disease, Acinetobacter baumannii accounts for about 80% of reported infections.

Medscape articles on infections associated with organisms mentioned in this lab exercise. Registration to access this website is free.

• Salmonellosis
• Typhoid fever
• Shigellosis
• Escherichia coli
• Proteusspecies
• Klebsiellaspecies
• Enterobacterspecies
• Serratiaspecies
• Yersinia enterocolitica
• Yersinia pseudotuberculosis
• Acinetobacter baumannii
• Pseudomonas aeruginosa
• Urinary tract infections
• Wound infections
• Community-acquired pneumonia
• Sepsis

SCENERIOS FOR TODAY'S LAB

Students will be assigned either Case Study 1A or 1B to do today. All students will do Case Study 2 as part of the results next time.

Case Study #1A

A 66 year old female with a history of recurring urinary tract infections and multiple antibiotic therapies presents with frequency and urgency of urination, dysuria, suprapubic discomfort, lower back pain, and a temperature of 99.2°F. A complete blood count (CBC) shows leukocytosis with a left shift. A urine dipstick shows a positive leukocyte esterase test, a positive nitrite test, 30mg of protein per deciliter, and red blood cells in the urine.

Assume that your unknown is a urine culture from this person.

Case Study #1B

A 72 year old female who is diabetic and a smoker was admitted to the hospital with a leg wound that is not healing. She appears confused and anxious, has a temperature of 102 °F , a heart rate of 101 beats per minute, a respiration rate of 29 breaths per minute, a blood pressure of 94/32 mm Hg, a urine output of only 110 cc for the last 8 hours, and a total white blood cell count of of 2300/µL. A blood culture is taken.

Assume that your unknown is a blood culture from this person.

CAUTION: TREAT EACH UNKNOWN AS A PATHOGEN!. Inform your instructor of any spills or accidents. WASH AND SANITIZE YOUR HANDS WELL before leaving the lab.

Taxo N® disk, alcohol, dropper bottle of distilled water, swab, and either a plate of MacConkey agar or a plate of Cetrimide agar, and an EnteroPluri-Test

PROCEDURE (to be done in groups of 3 )

[Keep in mind that organisms other than the Enterobacteriaceae and Pseudomonas can cause these infections, so in a real clinical situation other lab tests and cultures for bacteria other than those upon which this lab is based would also be done.]

1 . Perform a Gram stain on your unknown. Remember that the concentration of bacteria on slides prepared from taking bacteria off a petri plate tend to be much greater than those prepared by taking bacteria out of a broth culture, so be careful not to under decolorize. Continue decolorizing until the purple just stops flowing off of the bacterial smear, then wash with water.

Record the results of your Gram stain in the Gram stain section of Lab 13.

2. If you have a Gram-negative bacillus, determine if it is a fermentative Gram-negative bacillus like most Enterobacteriaceae or a non-fermentative Gram-negative bacillus such as Pseudomonas by performing an oxidase test as follows:

a. Using alcohol-flamed forceps, remove a Taxo-N® disc and moisten it with a drop of sterile distilled water.

b. Place the moistened disc on the colonies of the culture of your unknown.

c. Using a sterile swab, scrape off some of the colonies and spread them on the Taxo-N® disc.

In the immediate test, oxidase-positive reactions will turn a rose color within 30 seconds (see Fig. 10). Oxidase-negative will not turn a rose color (see Fig. 9). This reaction only lasts a couple of minutes. In the delayed test, oxidase-positive colonies within 10 mm of the Taxo-N® disc will turn black within 20 minutes and will remain black (see Fig 11). If the bacterium is oxidase-negative, the growth around the disc will not turn black (see Fig. 12).

Record your oxidase test results in the Oxidase test section of Lab 13.

3. If your unknown is oxidase-negative, indicating a fermentative Gram-negative bacillus, do the following inoculations:

a. Streak your unknown for isolation on a plate of MacConkey agar, a selective medium used for the isolation of non-fastidious Gram-negative rods and particularly members of the family Enterobacteriaceae, using one of the two streaking patterns illustrated in Fig. 4 and Fig. 5. Incubate upside down and stacked in the petri plate holder on the shelf of the 37°C incubator corresponding to your lab section.

b. Inoculate an EnteroPluri-Test as follows:

1. Remove both caps of the EnteroPluri-Test and with the straight end of the inoculating wire, pick off the equivalent of a colony from your unknown plate. A visible inoculum should be seen on the tip and side of the wire.

2. Inoculate the EnteroPluri-Test by grasping the bent-end of the inoculating wire, twisting it, and withdrawing the wire through all 12 compartments using a turning motion.

3. Reinsert the wire into the tube (use a turning motion) through all 12 compartments until the notch on the wire is aligned with the opening of the tube. (The tip of the wire should be seen in the citrate compartment.) Break the wire at the notch by bending. Do not discard the wire yet.

4. Using the broken off part of the wire, punch holes through the cellophane which covers the air inlets located on the rounded side of the last 8 compartments. Your instructor will show you their correct location. Discard the broken off wire in the disinfectant container.

5. Replace both caps and incubate the EnteroPluri-Test on its flat surface at 36°- 37°C for 18-24 hours.

4. If your unknown is oxidase-positive,indicating a non-fermentative Gram-negative bacillus, do the following inoculation:

a. Streak your unknown for isolation on a plate of Cetrimide agar, a selective and differential medium for Pseudomonas, using one of the two streaking patterns illustrated in Fig. 4 and Fig. 5. Incubate upside down and stacked in the petri plate holder on the shelf of the 37°C incubator corresponding to your lab section.

Note that MacConkey agar can also be used to isolate Pseudomonas but we are using the Cetrimide agar today because it enables us to detect the production of the blue to green water-soluble pigment by Pseudomonas aeruginosa, as well as the production of fluorescein.

You will also inoculate an EnteroPluri-Test for practice only, but keep in mind that the EnteroPluri-Test is used to identify Enterobacteriaceae, not Pseudomonas.

Case Study #2

After receiving a baby chicken for Easter, a 7 year old boy is taken to the emergency room with symptoms of vomiting, nausea, non-bloody diarrhea, abdominal cramps, and a temperature of 100°F. A complete blood count (CBC) shows the WBC count to be within the reference range.

This XLD agar plate and this EnteroPluri-Test are from a stool culture from this patient.

CAUTION: TREAT THE UNKNOWN AS A PATHOGEN!. Inform your instructor of any spills or accidents. WASH AND SANITIZE YOUR HANDS WELL before leaving the lab.

Demonstration XLD agar plate and EnteroPluri-Test

PROCEDURE (to be done in groups of 3 )

1. Observe the following demonstrations shown in the links directly below and identify the causative bacterium:

a. An XLD agar plate,a selective medium used for isolating and differentiating Gram-negative enteric bacteria, especially intestinal pathogens such as Salmonella and Shigella.

2. Record your results in the Results section of Lab 13.

C. Lab Tests Used as Part of Today's Lab

To isolate Enterobacteriaceae and Pseudomonas, specimens from the infected site are plated out on any one of a large number of selective and differential media such as EMB agar, Endo agar, Deoxycholate agar, MacConkey agar, Hektoen Enteric agar, and XLD agar. We will look at three of these.

1. MacConkey Agar

MacConkey agar is a selective medium used for the isolation of non-fastidious Gram-negative rods, particularly members of the family Enterobacteriaceae and the genus Pseudomonas, and the differentiation of lactose fermenting from lactose non-fermenting Gram-negative bacilli. MacConkey agar contains the dye crystal violet well as bile salts that inhibit the growth of most Gram-positive bacteria but do not affect the growth of most Gram-negatives (see Fig. 6).

If the Gram-negative bacterium ferments the sugar lactose in the medium, the acid end products lower the pH of the medium. The neutral red in the agar turns red in color once the pH drops below 6.8. As the pH drops, the neutral red is absorbed by the bacteria, causing the colonies to appear bright pink to red.

• Strong fementation of lactose with high levels of acid production by the bacteria causes the colonies and confluent growth to appear bright pink to red. The resulting acid, at high enough concentrations, can also causes the bile salts in the medium to precipitate out of solution causing a pink precipitate (cloudiness) to appear in the agar surrounding the growth(see Fig. 13).
• Weak fermentation of lactose by the bacteria causes the colonies and confluent growth to appear pink to red, but without the precipitation of bile salts there is no pink halo around the growth(see Fig. 15).
• If the bacteria do not ferment lactose, the colonies and confluent growth appear colorless and the agar surrounding the bacteria remains relatively transparent(see Fig. 17).

Typical colony morphology of our strains of Enterobacteriaceae and Pseudomonas aeruginosa on MacConkey agar is as follows:

1. Escherichia coli: colonies and confluent growth appear bright pink to red and surrounded by a pink precipitate (cloudiness) in the agar surrounding the growth (see Fig. 13). Strong fermentation of lactose.

2. Klebsiella pneumoniae: colonies and confluent growth appear bright pink to red but are not surrounded by a pink precipitate (cloudiness) in the agar surrounding the growth (see Fig. 14). Weak fermentation of lactose.

3. Enterobacter aerogenes: colonies and confluent growth appear bright pink to red but are not surrounded by a pink precipitate (cloudiness) in the agar surrounding the growth (see Fig. 15). Weak fermentation of lactose.

4. Enterobacter cloacae : colonies and confluent growth appear bright pink to red but are not surrounded by a pink precipitate (cloudiness) in the agar surrounding the growth (see Fig. 16). Weak fermentation of lactose.

5. Proteus mirabilis: colorless colonies agar relatively transparent (see Fig. 17). No fermentation of lactose.

6 . Proteus vulgaris: colorless colonies agar relatively transparent (see Fig. 18). No fermentation of lactose.

7 . Serratia marcescens : colorless colonies agar relatively transparent (see Fig. 19). No fermentation of lactose.

8 . Pseudomonas aeruginosa: colorless colonies agar relatively transparent (see Fig. 20).

9. Salmonella enterica: colorless colonies agar relatively transparent (see Fig. 21). No fermentation of lactose.

2 . XLD Agar

Xylose Lysine Desoxycholate (XLD) agar is used for isolating and differentiating Gram-negative enteric bacteria, especially intestinal pathogens such as Salmonella and Shigella. XLD agar contains sodium desoxycholate, which inhibits the growth of Gram-positive bacteria but permits the growth of Gram-negatives. It also contains the sugars lactose and sucrose, the amino acid L-lysine, sodium thiosulfate, and the pH indicator phenol red. Results can be interpreted as follows:

• If the Gram-negative bacterium ferments lactose and/or sucrose, acid end products will be produced and cause the colonies and the phenol red in the agar around the colonies to turn yellow(see Fig. 16).
• If lactose and sucrose are not fermented by the bacterium but the amino acid lysine is decarboxylated, ammonia, an alkaline end product will cause the phenol red in the agar around the colonies to turn a deeper red(see Fig. 17).
• Sometimes the bacterium ferments the sugars producing acid end products and breaks down lysine producing alkaline end products. In this case some of the colonies and part of the agar turns yellow and some of the colonies and part of the agar turns a deeper red(see Fig. 18).
• If hydrogen sulfide is produced by the bacterium as a result of thiosulfate reduction, part or all of the colony will appear black(see Fig. 19). Well-isolated colonies are usually needed for good results.

Typical colony morphology on XLD agar is as follows:

1. Escherichia coli: flat yellow colonies some strains may be inhibited.

2. Enterobacter and Klebsiella: mucoid yellow colonies.

3. Proteus: red to yellow colonies may have black centers.

4. Salmonella: usually red colonies with black centers.

5. Shigella, Serratia, and Pseudomonas: red colonies without black centers

Keep in mind, however, that some species and subspecies do not show typical reactions.

3. Cetrimide Agar (Pseudomonas P agar)

Cetrimide agar contains the chemical cetrimide (cetyl timethylammonium bromide) for the selective inhibition of most bacteria other than Pseudomonas. The medium also stimulates Pseudomonas aeruginosa to produce a number of water soluble iron chelators, including pyoverdin and pyocyanin. The green water soluble color characteristic of Pseudomonas aeruginosa is created when the yellow-green or yellow-brown fluorescent pyoverdin combines with the blue water-soluble pyocyanin (see Fig. 20). The fluorescent pyoverdin will typically fluoresce when the plate is placed under a short wavelength ultraviolet light (see Fig. 21). After a few minutes at room temperature, the plate loses its fluoresence. The fluoresence, however, can be restored by placing the plate back at 37°C for several minutes.

4. Oxidase Test

In this lab a Taxo N® disc is used to perform the oxidase test. The oxidase test is based on the bacterial production of an oxidase enzyme. Cytochrome oxidase, in the presence of oxygen, oxidizes the para-amino dimetheylanaline oxidase test reagent in a Taxo-N® disc.

• In the immediate test, oxidase-positive reactions will turn a rose color within 30 seconds(see Fig. 5). Oxidase-negative will not turn a rose color (see Fig. 6). This reaction only lasts a couple of minutes.
• In the delayed test, oxidase-positive colonies within 10 mm of the Taxo-N® disc will turn black within 20 minutes and will remain black(see Fig. 7). If the bacterium is oxidase-negative, the growth around the disc will not turn black (see Fig. 8).

Pseudomonas aeruginosa and most other non-fermentative, Gram-negative bacilli are oxidase-positive with the exception of the genus Plesiomonas, the Enterobacteriaceae are oxidase-negative.

5. Pigment production in Pseudomonas aeruginosa

The green water soluble color characteristic of Pseudomonas aeruginosa is created when the yellow-green or yellow-brown fluorescent pyoverdin combines with the blue water-soluble pyocyanin (see Fig. 20). The fluorescent pyoverdin will typically fluoresce when the plate is placed under a short wavelength ultraviolet light (see Fig. 21). After a few minutes at room temperature, the plate loses its fluoresence. The fluoresence, however, can be restored by placing the plate back at 37°C for several minutes. None of the Enterobacteriaceae produces pigment at 37°C.

6. Odor

Most of the Enterobacteriaceae have a rather foul smell Pseudomonas aeruginosa produces a characteristic fruity or grape juice-like aroma due to production of an aromatic compound called aminoacetophenone.

7. The EnteroPluri-Test

A number of techniques can be used for the identification of specific species and subspecies of Enterobacteriaceae. Speciation is important because it provides data regarding patterns of susceptibility to antimicrobial agents and changes that occur over a period of time. It is also essential for epidemiological studies such as determination of nosocomial infections and their spread.

In an effort to simplify the speciation of the Enterobacteriaceae and reduce the amount of prepared media and incubation space needed by the clinical lab, a number of self-contained multi-test systems have been commercially marketed. Some of these multi-test systems have been combined with a computer-prepared manual to provide identification based on the overall probability of occurrence for each of the biochemical reactions. In this way, a large number of biochemical tests can economically be performed in a short period of time, and the results can be accurately interpreted with relative ease and assurance.

The EnteroPluri-Test (see Fig. 22) is a self-contained, compartmented plastic tube containing 12 different agars (enabling the performance of a total of 15 standard biochemical tests) and an enclosed inoculating wire. After inoculation and incubation, the resulting combination of reactions, together with a Computer Coding and Identification System (CCIS), allows for easy identification. The various biochemical reactions of the EnteroPluri-Test and their correct interpretation are discussed below. Although it is designed to identify members of the bacterial family Enterobacteriaceae, it will sometimes also identify common biotypes of Pseudomonas and other non-fermentative Gram-negative bacilli. It does not identify Pseudomonas aeruginosa.

IDENTIFYING MEMBERS OF THE ENTEROBACTERIACEAE WITH THE ENTEROPLURI-TEST

The EnteroPluri-Test contains 12 different agars that can be used to carry out 15 standard biochemical tests (see Fig. 22). Interpret the results of your EnteroPluri-Test using the instructions below and record them on the EnteroPluri-Test table on your Results page. For more detail on the 15 biochemical tests in the EnteroPluri-Test, see Table 13A.

1. Interpret the results of glucose fermentation in compartment 1.

• Any yellow = + red = -
• If positive, circle the number 4 under glucose on your Results page.

2. Interpret the results of gas production also in compartment 1.

• White wax lifted from the yellow agar = + wax not lifted from agar = -
• If positive, circle the number 2 under gas on your Results page.

3. Interpret the results of lysine decarboxylase in compartment 2.

• Any violet = + yellow = -
• If positive, circle the number 1 under lysine on your Results page.

4. Interpret the results of ornithine decarboxylase in compartment 3.

• Any violet = + yellow = -
• If positive, circle the number 4 under ornithine on your Results page.

5. Interpret the results of H2S production in compartment 4.

• Black/brown = + beige = - (The black may fade or revert back to negative if the EnteroPluri-Test is read after 24 hours of incubation.)
• If positive, circle the number 2 under H2S on your Results page.

6. Indole production also in compartment 4. Do not interpret the indole test at this time. Add Kovac's reagent only after all other tests have been read (see step 16 below).

7. Interpret the results of adonitol fermentation in compartment 5.

• Any yellow = + red = -
• If positive, circle the number 4 under adonitol on your Results page.

8 . Interpret the results of lactose fermentation in compartment 6.

• Any yellow = + red = -
• If positive, circle the number 2 under lactose on your Results page.

9. Interpret the results of arabinose fermentation in compartment 7.

• Any yellow = + red = -
• If positive, circle the number 1 under arabinose on your Results page.

10. Interpret the results of sorbitol fermentation in compartment 8.

• Any yellow = + red = -
• If positive, circle the number 4 under sorbitol on your Results page.

11. Voges-Praskauer (VP) test in compartment 9. Do not interpret the VP test at this time. Add the reagents alpha-naphtol and potassium hydroxide (KOH) only after all other tests have been read (see step 17 below).

12. Interpret the results of dulcitol fermentation in compartment 10.

• Yellow = + green or dark brown = -
• If positive, circle the number 1 under dulcitol on your Results page.

13. Interpret the results of PA deaminase also in compartment 10.

• Dark brown= + green or yellow= -
• If positive, circle the number 4 under PA on your Results page.

14. Interpret the results of urea hydrolysis in compartment 11.

• Pink, red or purple = + beige = -
• If positive, circle the number 2 under urea on your Results page.

15. Interpret the results of citrate utilization in compartment 12.

• Any blue = + green = -
• If positive, circle the number 1 under citrate on your Results page.

16. Your instructor will add 2-3 drops of Kovac's reagent to the indole test compartment.

17. Your instructor will add 3 drops of alpha-naphtol reagent and 2 drops of potassium hydroxide (KOH) to the VP test compartment.

18. Add all the positive test number values in each bracketed section and enter each sum in its code box on the EnteroPluri-Test chart on your Results page.

19. The 5 digit number is the CODICE number. Look that number up in the Codebook and identify your unknown. (Should more than one organism be listed, the confirmatory tests indicated in the CCIS would normally then have to be performed. In addition, an identification of Salmonella or Shigella would usually be confirmed by direct serologic testing as will be described in Lab 17.)