30 December 2009

Clinical Trials

Medical Epidemiology Clinical Trials

A clinical trial is
* A cohort study
* A prospective study
* An interventional study
* An experiment
* A controlled study

The Structure of a Clinical Trial
Various Aspects Are Standardized and Protocol-based
* Subject selection (who are these people?!)
* Subject assignment
* H & P data
* Therapeutic intervention
* Lab calibration
* Outcome evaluation

Subject Selection
* Adequate number of subjects
* Adequate number of expected endpoints
* Easy to follow-up
* Willing to participate (give consent)
* Eligibility (criteria)
* Efficacy Versus Effectiveness
* Internal Validity (validity) versus External Validity (generalizability)

* Phase I: find toxic dose
* Phase II: no controls
* Phase III: RCT
* Phase IV: Post marketing?

Types of Control Groups
* Historical
* Contemporaneous
* Concurrent
* Randomized

Allocating Treatment
* Complete (Simple) randomization
* Restricted randomization

Complete Randomization
* Patients assigned by Identical chance process (but not necessarily in equal numbers)
* Mechanics
* Insures process fairness
* Does not insure balance, especially in small studies.Therefore, may still need statistical adjustment

* The only way to deal with unknown confounders.

Philosophy of Randomization
* Why are randomized trials not “epidemiologic” studies?
* Why randomization is so special?
* Has nothing to do with sampling bias.
* Randomization (random allocation) versus random sample.
* Does NOT deal with “chance” as a possible explanation of the difference. To the contrary.
* Can be used to create groups of unequal size.
* Baseline characteristics (table 1).

Allocation Concealment
* Define.
* Why do we need it?
* How is it done?
* Buzz words
* Versus blinding

Buzz Words
* Central (phone) randomization
* Sequentially numbered, opaque, sealed envelopes
* Sealed envelopes from a closed bag
* Numbered or coded bottles or containers

Restricted Randomization
* Stratification
* Blocking (Permuted Block Design)
* Stratified Blocking

Stratified Randomization
* Why

Scheme of stratified randomization
* Why?
* Ensures close balance of the numbers in each group at all times during trial.
* How is it done?
* More importantly when stratified.
* Problem If block size is discovered.
* Remedy: more blinding, varying block size, larger blocks.
* Basic, Randomized (random-sized), Stratified

Problems With Concurrent Controls

Use your imagination
Problems With Contemporaneous Controls
* Regional population differences.
* Regional practice differences.
* Diagnostic variations.
* Referral pattern biases.
* Variations in data collections.

Problems With Historical Controls
* A lot more

Why Do Controls in a Randomized Trial Do So Well ?!
* Volunteerism
* Eligibility
* Placebo effect
* Hawthorne effect
* Regression towards the mean

Placebo Effect
* Placebo can do just about anything (prolong life, cure cancer).
* Improve athletic performance
* Lower T4 count
* Placebo can do just about anything (prolong life, cure cancer).
* Placebo can also cause side effects (nocebo, Wile E Coyote effect).
* Placebo effect is very useful in medicine but in epidemiology it causes problems, so we try to equalize it between the 2 groups.
* We use placebo for other benefits.

Hawthorne Effect
* Hawthorne works of the Western Electric Co. Chicago, IL

Regression Towards the Mean
Course Evaluation Question
Explains difficult material:
* Strongly agree
* Agree
* Neutral
* Disagree
* Strongly disagree
* What difficult material ?

Regression Towards the Mean
Explains difficult material

Regression Towards the Mean

* Example
* Individuals with initially abnormal results tend on average to have more normal (closer to the mean) results later.
* Lab tests, BP etc.
* Recheck before randomization. Run-in period.
* Sophomore slump, medical school, Airforce landing feedback, most dangerous intersections.

Quicken Loans
* Rates this low seldom stay around long and tend to go up quickly and without warning

Why Does Prognosis Improve Over Time ?
1. Initial reports come from referral centers..
2. Publicity
3. Physicians’ awareness
4. Development of a Diagnostic Test
* Allows diagnosis of atypical cases.
* Is an incentive for physicians. It’s more challenging to diagnose difficult cases.
* Physicians with zero diagnostic skill can now diagnose this disease.
* Allows diagnosis of non-cases.
* Allows population based studies.
5. Publicity that a disease is very common relieves clinician from worrying that they may be overdiagnosing it.
6. Placebo effect increases over time. Why?

7. Safer treatment (laparoscopic cholecystectomy) lowers the threshold for doing surgery. So patients having surgery are not as sick as before.

Stage Migration Bias
Will Rogers Effect
8.Improved staging tests cause an apparent improvement of prognosis in every stage.
Stage Migration Bias Will Rogers Effect

Mortality by severity level
Severity distribution
Mortality by severity level
Will Rogers Effect
* When subjects with the most severe disease in each stratum are moved to the next (more severe) stratum - for whatever reason - this will cause an apparent improvement of prognosis in every stage.
* Common.
Exclusion Criteria
* Excluded patients are “ineligible”. So, Why the separate category?
* More informative
* Usually very large number.
* Usually underestimates. Real number even bigger. Why?

Exclusion Criteria
What to watch for
* Patient preference
* Clinician preference
* No reason given

Drop ins and Drop outs
* Define.
* Typical case
* Other

Subjects who drop out of study or change treatment. But available for outcome assessment.
* Intention to treat analysis
* Once randomized always analyzed
* Why ?

1. Change in therapy may be related to outcome or eligibility
2. To get the full benefit of randomization
3. Effectiveness versus efficacy
Five-Year Mortality in Coronary Drug Project


More on “Intention to Treat”
* Always analyze the results of the subjects according to the group they were randomized to. No exclusions.
* Even if they received no intervention.
* Even if they didn’t have the disease (example).
* The philosophy of “Intention to Treat” analysis
* Addresses the ultimate (and only) question for the clinician: Does prescribing treatment make a difference?
* LDL targets?!

Alternatives to “Intention to Treat” Analysis. (PROBLEMATIC)
* “Per Protocol” analysis.
How is it done?
* “As Treated” analysis.

“Per Protocol” analysis.
* Censoring data after subjects become non-adherent
* Preserves randomization
* Stops counting events (when? “Carry-over” effect)
* Reduced power

“As Treated” analysis
* Change the treatment arm of the subject as he/she changes exposure
* The follow-up time and the events will be assigned to current exposure
* Retain all events.
* Randomization violated.
* Have to assign “lag-time” (latency) and Carry-over time.

Loss to follow up
Differential vs. Random
+ Compare their baseline variables with the rest of the subjects.
+ Chase a subgroup.
+ Worst case scenario

Objectives of Subgroup Analysis
* Support the main finding
* Check the consistency of main finding
* Address specific concerns re efficacy or safety in specific subgroup

It may also generate hypotheses for future studies. But that is not a reason to do it.

Inappropriate Uses of Subgroup Analysis
* Rescue a negative trial
* Rescue a harmful trial
* Data dredging: find interesting results without a prespecified plan or hypothesis

To Avoid Inappropriate Uses of Subgroup Analysis
* Prespecify analysis plan.
* Prespecify hypotheses to be tested based on prior evidence.
* Plan adequate power in the subgroups
* Avoid the previous pitfalls.

Problems with Subgroup Analysis
* Low power
* Multiplicity
* Test for interaction
* Comparability of the treatment groups maybe compromised
* Over-interpretation

* PATIENTS Single blind.
* CLINICAL THERAPISTS usually double blind.
* Double Dummy
* CLINICAL EVALUATORS. Have to specify.
* Subjective vs. objective assessment

* Data and Safety Monitoring Board
* Have duty toward:
* Current study participants (ongoing treatment)
* Future participants.
* Enough evidence to change practice
* Enough evidence to withstand criticism. (Unable to randomize afterward).

Multiple looks
* Alpha spending
* Data Safety Monitoring Board
* Early Termination rules
* O’Brien-Fleming
* Early vs. late
* Benefit vs. harm (blinding?)
* Multiplicity
* Rules. Scenarios.
* Consecutive patients (versus a random sample)
* Baseline characteristics of patients (to see if randomization worked)
* Number of subjects and average duration of follow-up
(versus patient years)
* Interim analysis, problems
* Cumulative incidence (versus incidence density)
* Relative risk (Odds Ratio, or Hazard Ratio) (hopefully <1)is:
rate of outcome in a drug group
rate of outcome in a placebo group
* Relative risk reduction (similar to attributable risk %). But here it is 1-RR.
* Absolute difference in risk (ADR)= risk in control group – risk in intervention group (similar to AR) very important, sometimes not reported
* Relative risk reduction versus absolute difference in risk
* Number needed to treat NNT =1/ADR very useful (remember time)

Descriptions of “Trials”
* 34% relative decrease in the incidence of MI. The decrease is statistically significant. The 95% CI ranges from 55% relative decrease to a 9% relative decrease.

* 1.4% decrease in …. (2.5% versus 3.9%). The decrease is statistically significant. The 95% confidence interval ranges from a 2.5% decrease to a ..

* 77 persons must be treated for an average of just over 5 years to prevent 1 MI.

Ratings of Trials
(-5=harmful,+5=very effective)

Medical Epidemiology Clinical Trials.ppt


29 December 2009



1. Random collection taken at any time of day with no precautions regarding contamination. The sample may be dilute, isotonic, or hypertonic and may contain white cells, bacteria, and squamous epithelium as contaminants. In females, the specimen may cont contain vaginal contaminants such as trichomonads, yeast, and during menses, red cells.
2. Early morning collection of the sample before ingestion of any fluid. This is usually hypertonic and reflects the ability of the kidney to concentrate urine during dehydration which occurs overnight. If all fluid ingestion has been avoided since 6 p.m. the previous day, the specific gravity usually exceeds 1.022 in healthy individuals.
3. Clean-catch, midstream urine specimen collected after cleansing the external urethral meatus. A cotton sponge soaked with benzalkonium hydrochloride is useful and non-irritating for this purpose. A midstream urine is one in which the first half of the bladder urine is discarded and the collection vessel is introduced into the urinary stream to catch the last half. The first half of the stream serves to flush contaminating cells and microbes from the outer urethra prior to collection. This sounds easy, but it isn't (try it yourself before criticizing the patient).
4. Catherization of the bladder through the urethra for urine collection is carried out only in special circumstances, i.e., in a comatose or confused patient. This procedure risks introducing infection and traumatizing the urethra and bladder, thus producing iatrogenic infection or hematuria.
5. Suprapubic transabdominal needle aspiration of the bladder. When done under ideal conditions, this provides the purest sampling of bladder urine. This is a good method for infants and small children.

Full details here


28 December 2009

Clinically Relevant Microbiology Starts at the Source

Clinically Relevant Microbiology Starts at the Source
By: Mike Costello, PhD, MT(ASCP)
ACL Laboratories
Mary Dikeman, MT (ASCP)
Affinity Health System

Program Objectives
* Emphasize that obtaining sensitive and specific microbiology results begins with the patient and not at the door of the microbiology laboratory.
* Accentuate the importance of proper collection and transport of specimens in both local and referral environments
* Stress the importance of timely communication between the Microbiology laboratory and those collecting specimens
* Describe common pitfalls in specimen collection and transport
* Discuss What rules or principles must be followed in order to collect microbiology specimens which will accurately reflect the pathogenesis of the microbiological agent. (Church D. The Seven Principles of Accurate Microbiology Specimen Collection. . Calgary Laboratory Services Microbiology Newsletter. Volume 6, 2005)

The practice of sensitive, specific and cost effective clinical microbiology is intimately tied to the submission and proper handling of optimal specimens for analysis. Unfortunately, these aspects of clinical microbiology are not as critically controlled as our laboratory assays. It is our responsibility to educate and notify our healthcare colleagues when specimens arrive at the laboratory that will yield inferior results.

Quality assurance of specimen collection and transport is a never ending battle and requires long term commitment of your time and resources, but the end results are better patient care and a more rewarding experience for those of us who work in the microbiology laboratory.

Principle #1: The specimen must be collected with a minimum of contamination as close to
site of infection as possible

Urine Culture Contamination Rates

* Urine Culture contamination rates (>2 bacteria at >100,000 CFU) should be <20%
o CAP Q-Probe study (Valenstein P Meier F. Urine culture contamination: a College of American Pathologists Q-Probes study of contaminated urine cultures in 906 institutions. Arch Pathol Lab Med. 1998;122:123-129)..
+ 630 participants collected information of 155,037 urine culture specimens; 20.1% were considered contaminated (>2 organisms at >105 CFU)
+ The top 10% of institutions reported a rate of 5.6%. Bottom 10% of institutions reported a contamination rate of 36.8%
+ Males have a lower contamination rate than females (11.2% Vs. 22.8%)
+ ER departments had a contamination rate of 17.8%, sites adjacent to lab had rates of 19.5%, and other sites had rates of 22.1%

Blood Culture
* Two sets of blood cultures should be drawn. Number of sets positive correlates with true sepsis (except for coagulase negative Staph?) (Clin Microbiol. Rev 19:788-802, 2006)
* Catheter drawn blood cultures
o Catheter drawn blood cultures are equally likely to be truly positive (associated with sepsis), but more likely to be colonized (J Clin Microbiol 38:3393, 2001.)
+ One drawn through catheter and other though vein PPV 0f 96%
+ Both drawn from catheter PPV 0f 50%
+ Both drawn through vein PPV of 98%
o Study of positive coagulase negative Staphylococcus cultures and sepsis (Clin Infect Dis. 39:333, 2004.)

Blood Culture Contamination Rate
By Service Drawing Culture
What is an “Acceptable” Blood Culture Contamination Rate for Your Lab??
Blood Culture Contamination in Pediatric Patients
Young Children and Young Doctors
Inexperienced physician-young child
Inexperienced physician-older child
Experienced physician-younger child
Experienced physician-older child
Predicative Value of a Positive Result
False Positive
True Positive
Ped Infect Dis. 2006, 25:611-614.

Inexperienced Physicians = Interns and residents in 1st half of training
Experience Physicians = Residents in 2nd half of training and senior physicians
What is an “Acceptable” Blood Culture Contamination Rate for Your Lab??

What is an “acceptable” blood culture contamination rate*?
Berkeris LG, JA Toworek, MK Walsh, PN Valenstein. Trends in Blood Culture Contamination.
Arch Pathol Lab Med 129:1222-1294, 2005

Respiratory Cultures
* Community Acquired Pneumonia – Sputum rejection rate and culture correlation with gram stain
o 54% of all samples were judged to be of good quality.
o Presence of a (predominant morphology) PM on Gram stain was predictive of whether the sputum culture could demonstrate a pathologic organism. In the presence of a positive PM, 86% of cultures yielded a pathologic organism, while a positive culture was obtained in 19.5% of Gram stains without a predominant organism. S. pneumoniae was the most common infection, growing in 55.7% of positive sputum cultures.
o The sensitivity and specificity of finding Gram-positive diplococci for a positive culture of S. pneumoniae were 60% and 97.6%, respectively (Arch Intern Med. 2004;164:1725-1727, 1807-1811)
* Ventilator associated pneumonia (VAP) – appropriate specimen
o Blood cultures highly specific but not sensitive (positive in <10% of VAP)
o Quantitative cultures of lower respiratory tract specimens show a closer clinical correlation than sputum subcultures (Clinical Microbiol. Rev. 19:637-657, 2006.)

Viral Respiratory Cultures – Collect Sample From Site of Infection
How do you know that an adequate
Specimen was submitted for rapid
EIA assays???
Throat swabs are even worse!
Samples for Diagnosis of Viral Respiratory Infections
Lung biopsy
Bronchial alveolar
Nasopharygeal secretion
Nasopharygeal wash
Induced sputum
Nasopharygeal swab
Nasal wash

Throat swab (adenovirus only)
LRTC cells absent
Reagent Cost

Skin and Soft Tissue (Wound) Cultures
* Collect with steel (needle aspirate or scalpel)
* Discourage the use of swabs
* If infection NOT suspected, DON’T culture
* Get infected tissue or body fluid [ discourage swabs! ]
* -use something sharp ( syringe, scalpel, etc )
* -close doesn’t count
* *Don’t culture the surface / get deep infected sample*
* Remove needles / send capped syringe with aspirate
* Share specimen: Microbiology-Surgical Path-Cytology
* ** Label specimen and site accurately
* ** Give appropriate history
(Matkoski C. Sharp SE, Kiska DL. Evaluation of the Q Score and Q234 Systems for cost-effective and clinically relevant interpretation of wound cultures. J Clin Microbiol 2006;44:1869-1872)

Principle #2: A specimen must be collected at the optimal time(s) in order to recover the pathogen(s) of interest
Principle #3: A sufficient quantity of the specimen must be obtained to perform the requested tests
Blood Cultures
* Volume of blood drawn is the single most important factor influencing sensitivity. A single set for an adult blood culture consists of one aerobic and one anaerobic bottle. Optimally 10 mL of blood should be inoculated into each bottle. Volume of blood for a pediatric culture can be related to the infants weight
* Solitary blood cultures should be less than 5% (Arch Pathol Lab Med. 2001 125:1290-1294)
* If only enough blood can be drawn for one bottle, inoculate the aerobic bottle.
o 644 positive blood cultures, 59.8% from both bottles, 29.8% from aerobic bottle only and 10.4% from anaerobic bottle only (J Infect Chemother 9:227, 2003).
Pediatric Blood Cultures - Volume
Surgical Specimens (Other Shared Specimens)
Specimen size of pea or larger
Anaerobic transport tube
insert specimen and recap
and stain

Acceptable Specimens For Anaerobic Culture

Principle #4: Appropriate collection devices and specimen containers must be used to ensure recovery of all organisms
Recovery of Anaerobic Bacteria Placed in in Aerobic/Anaerobic Transport Media
CVP = Copan Vi-Pak Amies Agar Gel collection and transport swabs
SSS = Starplex StarSwab II,
PAC = BBL Port-A-Cult

How Does Transport Time Affect Yields?
J Clin Microbiol. 2001:39 377-380

Suggested Transport Media – General Comments

Principle #5: Collect all microbiology test samples prior to the institution of antibiotics
Principle #6: The specimen container must be properly labeled and sealed prior to transport
Principle #7: Minimize transport time or maximize transport media. There is always some loss of viability during transport
Minimize transport time and maximize use of transport media as much as possible
Environmentally Fragile Organisms
QA monitor??
Principle #8: Special handling/Collection instruction must be followed
* First, communicate with those that are doing collections.
* Collection instructions are written and available.
* Get involved with nursing orientation/education days and ask to have the instructions given out; poster board learning; quiz or competencies.
* Talk to providers when there are problems with specimen collection; they sometimes do not know they could do it better.

Principle #9: Improper specimen Collected for Ordered Test

Criteria For Rejection of Microbiological Specimens
* Criteria for rejection must be readily available and laboratory specific
* Unlabeled or improperly labeled specimen
* Prolonged storage or transport
* Improper or damaged container
* Specimen received in fixative
* Oropharyngeal contaminated sputum
* Duplicate specimens stools, sputum) within a 24 hour period. Exceptions cleared by the laboratory
* Specimens unsuitable for culture request (anaerobic culture from not acceptable source, urine from Foley catheter)
* Dry Swab
* 24-hr collection of urine or sputum for AFB or fungal culture
* Other criteria specific to your laboratory

Cultures That Should Include a Gram Stain
* CSF or sterile body fluid (cytospin)
* Eye
* Purulent discharge
* Sputum or transtracheal aspirate
* All surgical specimens
* Tissue
* Urethral exudates (male only, intracellular gonococcus))
* Vaginal specimens
* Wounds

* Publish specific rules for specimen collection
o There will be exceptions!
+ Make physician or healthcare provider aware of implications of culturing suboptimal specimens
* Communicate, communicate, communicate!
o Real time feedback
o Contact the health care worker who collected the suboptimal specimen


* Clinical Microbiology Procedures Handbook. 2nd Edition. . HD Isenberg ed. ASM. Cumitechs. ASM Press. Wash. DC.
* Manual of Clinical Microbiology, 9th Edition. ASM Press. Wash. DC. 2007.Miller MJ.
* A Guide To Specimen Management in Clinical Microbiology. ASM Press. Wash. DC. 1999.

Clinically Relevant Microbiology Starts at the Source.ppt


Specimen Collection and Laboratory Diagnosis of Lower Respiratory Infections

Specimen Collection and Laboratory Diagnosis of Lower Respiratory Infections

By:Mohammad Rahbar (PhD)
Department Of Microbiology Reference Laboratory of Iran

Anatomy of Respiratory Tract

“ The culture of lower respiratory specimens may result in more unnecessary microbiologic effort than any other type of specimen.”
Raymond C Bartlett

Lower Respiratory Tract Infections
* Pneumonia is the sixth leading cause of death in US
* Increasing numbers of patients at risk
o Aging population
o Increase in patients with immunocompromising conditions
* Overtreatment has lead to resistance
o Multidrug resistant Streptococcus pneumoniae
o Resistance among hospital acquired pathogens such as Acinetobacter, Pseudomonas aeruginosa E.coli K.pneumonia (ESBLs) MRSA and others
* Major sections
o Clinical aspects of diseases of LRT
o Specimen collection
o Specimen processing
o Interpretation of bacterial cultures
o Most common pathogens
o Methods for implementing change
o Guidelines for frequency of testing
o Public health issues
o Reimbursement codes

Categories of Lower Respiratory Tract Infections
* Acute bronchitis
* Community acquired pneumonia
* Hospital acquired pneumonia
* Pneumonia in the immunocompromised host

Community Acquired Pneumonia Etiologic Agents
Community Acquired Pneumonia Diagnosis

Available Test Methodologies
* Sputum Gram stain and culture
* Blood cultures
* Serologic studies
* Antigen detection tests
* Nucleic acid amplification tests

Sputum Gram Stain and Culture
* Demonstration of predominant morphotype on Gram stain guides therapy
* Accuracy is good when strict criteria are used
* Cheap, so why not?

* Poor specimen collection
* Intralaboratory variability (Gram stain interpretation)
* Low sensitivity and specificity
* Empiric treatment guidelines
* Not cost effective

Sputum Collection
* Proper patient instruction
o Food should not have been ingested for 1-2 h prior to expectoration
o The mouth should be rinsed with saline or water
o Patient should breathe and cough deeply
o Patient should expectorate into a sterile container
* Transport container immediately to lab
* Perform Gram stain and plant specimen as soon as possible

Sputum collection
* Sputum of less than 2ml should not be processed unless obviously purulent
* Only 1 sputum per 24hr .submitted
* Some scoring system should be used to reject specimen that re oral contamination.
* Transportation in <2 hr is recommended with refrigeration if delays anticipated.
* Handle all samples using universal precautions.
* Perform Gram stain and plant specimen as soon as possible

Induced sputum
Patients who are unable to produce sputum may be assisted by respiratory therapy technician. Aerosol induced specimen are collected by allowing the patient to breath aerosolized droplets of a solution of 15% sodium chloride and 10% glycerin for approximately 10 minute . obtaining such specimen may avoid the need for a more invasive procedures ,such as bronchoscopy or needle aspiration, in many cases.

Gastric aspiration
* The gastric aspiration is used exclusively for isolation of acid-fast bacilli and may be collected from patients who are unable to produce sputum, particularly young children. The relative resistance of mycobacteria allows them to remain viable for a short period. Gastric lavage must be delivered to the lab immediately so that the acidity can be neutralized. Specimen can be first neutralized and then transported if immediate delivery is not possible.

Sputum Gram Stain Unacceptable
Sputum Gram Stain Good Quality
Good quality specimens

* Quantify number and types of inflammatory cells
* Note presence of bronchial epithelial cells
* Concentrate on areas with WBCs when looking for organisms
* Determine if there is a predominant organism (> 10 per oil immersion field)
o Semiquantitate and report organism with descriptive
o If no predominant organism is present, report “mixed gram positive and gram negative flora”

Utility of the Gram Stain in Diagnosis of Pneumonia
Roson, B, et. al. 2000. Clin Infect Dis 31:869-74.

* Prospective study
* Non immunocompromised patients hospitalized with CAP
* 1,000 bed hospital in Spain
* ER physicians instructed on sputum collection for Gram stain and culture
* Sputum collected under supervision of nurse or resident
* Sputum collected under supervision of nurse or resident
o Samples were processed immediately
o Screened for epithelial cells
o Screened for predominant morphotype (> 75% of the organisms seen)
o Sputum planted to blood agar, chocolate agar and MacConkey agar
* Strictly defined clinical and diagnostic parameters

Utility of the Gram Stain in Diagnosis of Pneumonia
Roson, B, et. al. 2000. Clin Infect Dis 31:869-74

* 190/533 (35.6%) patients had no sputum sample submitted (these patients were included in the calculations)
* 133/533 (25%) patients had a poor quality specimen
* 210/533 (39.4%) patients had a good quality specimen
* Overall sensitivity and specificity for pneumococcal pneumonia: 57% and 97%
* Overall sensitivity and specificity for H. influenzae pneumonia: 82 % and 99%
* Gram stain gave presumptive diagnosis in 80% of patients who had a good specimen submitted
* > 95% of patients in whom a predominant morphotype was seen on Gram stain received monotherapy

Gram Stain Reports
* Be as descriptive as possible
o Moderate neutrophils
o Moderate Gram positive diplococci suggestive of Streptococcus pneumoniae
o Few bacteria suggestive of oral flora
* Keep report short—avoid line listing of all morphotypes present

Sputum and Endotracheal Suction Culture Evaluation
* Identify and perform susceptibility testing on 2-3 potential pathogens seen as predominant on Gram stain
* Alpha strep—rule out S. pneumoniae
* Yeast—rule out Cryptococcus neoformans only
* S. aureus, Gram negative bacilli
o < normal flora, quantify and limit ID; no susceptibility
o Add comment that organism not predominant on stain
* ID mould, Mycobacteria or Nocardia spp.

IDSA Practice Guidelines
Diagnostic Tests for CAP
* Outpatients
o Empiric therapy with a macrolide, doxycycline, or a fluoroquinolone
* Hospitalized patients with CAP
o Gram stain and culture of sputum
o 2 pretreatment blood cultures
o Studies for Mtb, Legionella in select patients
Bartlett JG. 2000. Clin Infect Dis 31:347-82.
* Rationale
o To improve patient care
o Advance knowledge of epidemiologically important organisms
o Prevent antibiotic abuse
o Reduce antibiotic expense
Bartlett JG. 2000. Clin Infect Dis 31:347-82.

ATS Guidelines Diagnostic Tests for CAP
* Empiric therapy for outpatients
o Macrolide or tetracycline
* Hospitalized patients with CAP
o 2 sets of pre-treatment blood cultures
o Pleural fluid Gram stain/culture when appropriate
o Studies for Legionella, Mtb, fungi in select patients
o Sputum Gram stain/culture only if resistant or unusual pathogen is suspected
o Avoid extensive testing
ATS. 2001. Am J Respir Crit Care Med 163: 1730-1754.

Hospital Acquired Pneumonia
* Most frequent nosocomial infection (30-33% of cases) among combined medical surgical intensive care units
* 83% are ventilator associated
* Etiologic agents Frequency (%)
o Gram positive cocci
+ S. aureus 17
+ S. pneumoniae 2-20

* Aerobic gram-neg bacilli 60
o Pseudomonas aeruginosa
o Enterobacter sp.
o Klebsiella pneumoniae
o Acinetobacter
o Legionella
o Anaerobes 10-20
o Fungi 0-10
Modified from: Carroll KC. 2002. J Clin Microbiol 40: 3115-3120.

Hospital Acquired Pneumonia Diagnosis

* American College of Chest Physicians: Clinical findings are not sufficient for definitive diagnosis
* Qualitative culture or endotracheal sputum has poor predictive value
* Bronchoscopy is recommended by many pulmonologists
o Bronchial brushings
o Bronchial washes
o Protected specimen brushing
o Bronchoalveolar lavage specimens (BAL)
o Transbronchial biopsy

Respiratory Specimens
* Protected Brush Specimen
o To procure uncontaminated lower airway secretions
o Brush within 2 catheters
* Bronchoalveolar Lavage (BAL)
o Samples large area of the lung
o Performed using a bronchoscope
o 100 to 250 ml of saline injected
o Injected saline along with secretions is collected by aspiration
* Transthoracic Aspiration
o Involves percutaneous introduction of a needle directly into the infiltrate

Bronchoalveolar Lavage (BAL) Specimen Acceptability
* Microscopic examination of Gram-stained smear
o Acceptable
+ <1% of cells present are squamous epithelial cells
o Unacceptable
+ >1% of cells present are squamous epithelial cells
Thorpe JE et. al. 1987. Bronchoalveolar lavage for diagnosing acute bacterial
pneumonia. J. Infect. Dis. 155:855-861

Processing Bronchoscopy Specimens
* Bronchoscopy brush protected
o Aerobic bacterial culture and Gram stain
o Anaerobic bacterial culture
o Limited volume
* Bronchoscopy brush, unprotected
o No anaerobic culture
o Limited volume
* Bronchial washings
o Useful only for pneumonia caused by strict pathogens
o Reasonable requests: Mtb, Fungi, Legionella, Pneumocystis
* Bronchoalveolar lavage
o No anaerobe culture
o Amenable to extensive testing for all opportunistic pathogens

Interpretation of Quantitative PSB/BAL
* Dilution Method
o Quantify each morphotype present and express as CFU/ml
* Calibrated Loop Method
o Quantify each morphotype present and express as log10 colony count ranges
* Thresholds for significance
o PSB > 103 CFU/ml
o BAL > 104 CFU/ml

Bronchoscopy Samples Quantitative Methods
Routine culture
* Most of the commonly sought etiologic agents of lower respiratory tract infection will isolated on routinely used media : 5% sheep blood agar ,MacConkey agar for isolation and differentiation of gram-negative bacilli ,and chocolate agar for Neisseria spp and Haemophilus
* Because of contaminating oral flora ,sputum specimens ,specimens obtained by bronchial washing, and lavage trachestomy, or endotracheal tube aspirates are not inoculated to enriched broth or incubated anaerobically. Only specimens obtained by percutaneous aspiration (including transtracheal aspiration )and by protected bronchial brush are suitable for anaerobic culture: he latter must be done quantitatively for proper interpretation.
* Transtracheal and percutaneous lung aspiration material may be inoculated to enriched thioglycollate ,as well as to solid media. For suspected cases of legionnaires disease buffered charcoal –yeast extract (BCYE) agar and selective BCYE are inoculated.

* Sputum specimens from patients known to have cystic fibrosis should be inoculated to selective agar ,such as manitol salt agar for recovery of S .aureus and selective horse blood-bacitracin ,incubated anaerobically and aerobically ,for recovery of H,influenzae that may be obscured by the mucoid P,aeroginosa on routine media. The use of selective medium for B.cepacia ,such as PC or OFPBL agar ,is also recommended

Immunocompromised Patients
Suggested BAL Protocol
* Aerobic Gram stain quantitative bacterial culture
* Fungal stain and culture
* Mycobacterial stain and culture
* Viral culture/Respiratory DFA
* Pneumocystis DFA
* Legionella culture

Specimen Collection and Laboratory Diagnosis of Lower Respiratory Infections.ppt


Obtaining Specimens for Microbiological Evaluation

Obtaining Specimens for Microbiological Evaluation

Bacteremia I
* Most bacteremias are intermittent
* One blood culture is rarely sufficient
o Staphylococcus epidermidis
+ Frequent contaminant
+ Commonest cause of PVE
* Two blood cultures usually sufficient
o Three or four if suspect likely contaminant
o Antibiotic therapy

Blood Cultures - Volume
The magnitude of bacteremia may be low (<1cfu/ml)

Higher volumes have higher yield
Blood Cultures - Lab Aspects
* Additives (SPS, resins) increase yield
* Aerobic and anaerobic bottle = one blood culture
* Five days incubation sufficient
o Exception: Brucella, Histoplasma, Mycobacterium, Bartonella, Legionella
* Automated Systems detect CO2
o Subculture detected bottles

Aerobic/Anaerobic Blood Culture Bottles
AFB Blood Culture Bottle
Obtaining Blood Culture
* Locate the vein
* Prep kit
o Alcohol 5 sec. Dry 30-60 sec
o Tincture of Iodine-center to periphery. Dry 45-60 sec
* Remove caps, clean with alcohol
* Put on gloves
* Without palpating, draw 20 ml and put 10 in anaerobic and 10 in aerobic bottle
* Dispose of syringe in sharps container
* Label bottles and send to lab

Blood Culture Prep Kit
Sputum Culture Reliability
* Expectorated unreliable because of contamination
o Reliability  if physician observes
* Laboratory reliability screen
o > 25 PMN’s, < 10 oral squamous cells per hpf

Sputum Container
* Gram stain
o Useful for immediate therapy
o May be more reliable than culture
+ Many PMN’s with single bacterial morphology
* AFB - first morning specimen
* Pneumocystis carinii - induced specimen

Nasal Cultures
* Virus
o Use wire swab
o Place in nose 1-3 cm, rotate, 10-15 sec
o Obtain viral transport medium from lab
* Bacterial
o Culturette with rigid or wire swab
o Suspect pertussis - special media

Wire Swab
Throat Cultures
* For Group A strept, diphtheria, gonorrhea
* Tongue blade - visualize pharynx and tonsils
* Rub swab over tonsils and pharynx
* Insert into holder, crush vial

Swabs for Bacterial (red) and Viral (green) Cultures

Cerebrospinal Fluid
* Use sterile technique
* First or second tube to Microbiology
* Studies
o Gram stain - one drop cloudy fluid or sediment
o Aerobic culture - 1.0 ml
o Viral culture - 1.0 ml
o AFB or fungal culture - up to 10 ml

Wounds: General Principles
* Closed space infections provide reliable specimens
* Open wounds heavily contaminated
o May quantitate
* May obtain culture by aspirating advancing border
* Culture skin, soft tissue or wound abscesses for anaerobic and aerobic organisms
o Transport in capped syringe or special tube

Wound Culture
* Closed space abscesses
o Decontaminate skin
o Insert needle and aspirate or aspirate pus after incision
* Open wound
o Remove superficial exudate
o Aspirate through margin or swab (least reliable)
* Transport
o Capped syringe or anaerobic transport tube
o Rapidly to lab

Urine - General
* Collection method must avoid contamination
o Clean catch, midstream voided
o Catheterized urine
o Suprapubic aspiration
* Cultures performed quantitatively
o Less than 10,000 per ml suggest contamination

Clean Catch, Midstream Urine
* Cleanse periurethral area with soap and water
* Pass initial urine into toilet, then collect specimen in cup
* Instructions to patient are critical

Instructions for Patient
* Remove underpants completely so they will not get soiled.
* Sit comfortably on the seat, but do not leave your knees in front of you. Instead swing one knee to the side as far as you can.
* Spread yourself with one hand, and continue to hold yourself spread while you clean and collect the specimen.
* Wash—Be sure you wash well and rinse well before you collect your urine sample. Wash only the area from which you pass urine. You do not have to wash hard, but wash slowly. Be sure to wipe from the front of your body towards the back. Wash between the folds of skin as carefully as you can.
* Do not put sponges in the toilet. Put them back in the plate.
* Rinse—After you have washed with each soap pad, rinse with each moistened pad with the same front to back motion. Do not use any pad more than once.
* Hold cup by the outside and pass your urine into the cup. If you touch the inside of the cup or drop it on the floor, ask the nurse to give you a new one.

Catheterized Urine
* Cleanse periurethral area with soap and water
* Insert catheter into bladder
o Discard initial urine
o Collect specimen in sterile cup
* Chronic indwelling Foley catheter
o Clamp tubing below junction (or port)
o Disinfect with alcohol
o Insert needle (on syringe) through port or catheter wall and aspirate

Suprapubic Aspiration
* Prep skin with alcohol or iodine
* Anesthetize with lidocaine
* Introduce needle 2.0 cm above symphysis
* Aspirate 20 ml for culture

Suprapubic Aspiration
Wire Swab

Obtaining Specimens for Microbiological Evaluation.ppt


Specimen collection Role of the Nurse

Specimen collection Role of the Nurse

Nurses often assume the responsibility of specimen collection

* Specimens consist
o Urine
o Stool
o Sputum
o Wound drainage
o Blood

What about the client?

* Comfort
* Privacy
* Questions
* Clear, concise directions

The Nurse

* Check physician orders
* Keep it Simple directions to client
* Standard precautions
* Label specimen
* Timely
* C&S to lab ASAP or refrigerated
* Documentation

Urine Specimen

* Random
* Clean
* Female ? Menses (make note)
* Tested for:
+ Specific gravity
+ pH
+ Albumin
+ Glucose
+ Microscopic exam

Urine for C&S

* Culture = ? Bacteria growing
* Sensitivity = which antibiotics are effective
* Readings after 24; 48; 72 hrs.

Midstream Urine

Sterile Catheter Specimen

(never from bag)

Why a urine specimen for C&S

* ? Urinary Tract Infection (UTI)
o Frequency
o Urgency
o Dysuria
o Hematuria
o Flank pain
o Fever
o Cloudy, malodorous urine

Obtaining specimen

* Wash hands
* Clean meatus, female front to back
* Start stream, then stop, collect specimen
* Aseptic technique
* Bedpan/mexican hat
* To lab 15-20min post collection


* Pediatric bags ( u Bag)
* Never squeeze diaper

Characteristics of Urine

* Color
* Clarity
* Odor

Specimen Collection

* Random Specimens
o Clean-not sterile
o Ordered for
+ Urinalysis testing
+ Measurement of specific gravity
+ pH
+ Glucose levels

Urine specimen collection

* Midstream Specimen
o Clean voided
o C&S
o 30-60 mls urine
* Sterile Specimen
o Indwelling catheter
o Drainage bag

Urine collection

* Timed urine specimens
o 2-72 hr intervals (24hr most common)
o Begin after urinating
o Note start time on container & requisition
o Collect all urine in timed period

Post Reminder Signs

Indwelling Catheter

* Strict aseptic technique
* Only from Bag if Brand new
* Sampling Port?
* Clamp 30 min. prior
* Wash hands – Glove
* Cleanse port with alcohol swab
* Sterile needle
* To lab 30 min (may refridge 2hrs)

Common Urine Lab Tests

* Routine Urinalysis
o Examine within 2hrs
o 1st voided specimen in AM
o Reagent strip
* Specific Gravity
o Concentration
o 1.010-1.025
* Urine glucose
o Diabetics
o Reagent strips
o Double void

Measuring chemical properties of urine=Urinalysis

* Glucose
* Ketones
* Protein
* Blood- hematuria
* pH
* Specific gravity
* Microscopic examination

Stool Specimen

Analysis of fecal material can detect pathological conditions ie: tumors, hemorrhage, infection

* Tests
o OB
o Pus
o Ova & Parasites

Fecal specimens

* ? Chemical preservatives
* Medical aseptic technique
* To lab on time
* Labelling
* Documentation

Guaiac Test

Colorectal cancer screening test


Hemoccult slide test

Fecal Characteristics

* Color
o melena
* Odor
* Consistency
* Frequency
* Amount
* Shape
* Constituents

Guaiac Test

* Single positive test result does not confirm bleeding or colorectal cancer.
* Repeat test 3X
* Meat free, high residue diet

Vaginal or Urethral Discharge Specimens

* Normally thin, nonpurulent, whitish or clear, small in amount
* S&S STD’s, UTI
* Not Delegated
* Assess external genitalia
* If STD record sexual history
* Physician’s order- vaginal/urethral

Blood Specimens

* Lab techs
* ABG’s
* Blood Glucose

Respiratory Tract

* Tests to determine abnormal cells or infection
o Throat cultures
o Sputum specimens
o Skin testing
o Thoracentesis

Nose, Throat Specimens

* Upper respiratory/ throat infections
* Should Not be delegated
* Throat swabs
o ac meal or 1 hr pc meal
o Wash hands, glove
o Tilt head backward
o “ah” ( if pharynx not visualized, tongue depressor, anterior 1/3 of tongue)
o Don’t contaminate

Throat cultures

* Oropharynx & tonsillar
* Sterile swab
* Culture determines pathogenic microorganisms
* Sensitivity determines the antibiotics to which the microorganisms are sensitive or resistant

Method for throat culture

* Insert swab into pharyngeal region
* Reddened areas/ exudate
* Gag reflex if client sitting and leaning forward slightly
* Inform client re procedure

Nose culture

* Blow nose, check nostril patency
* Rotate Swab inflamed mucosa or exudate
* Swab must advance into nasopharynx to ensure culture properly obtained

Sputum specimens (3 major types)

Ordered to identify organisms growing in sputum

* C&S
o 3 consecutive, early am
* Cytology
o Abnormal lung cancer by cell type
o 3 early am

Sputum collection

* May be delegated
* Cough effectively
* Mucus from bronchus
* Not Saliva
* Record
o Color
o Consistency
o Amount
o Odor
o Document date & time sent to lab.

Sputum collection

* No mouthwash/toothpaste-

viability of microorganisms and alter culture results

Skin testing

* Determines pulmonary diseases
o Bacterial
o Fungal
o Viral

Antigen injected intradermally

Injection site circled

Instructions not to wash site

Reading skin test

* Induration – palpable, elevated, hardened area around site. Edema and inflammation from antigen –antibiotic reaction. Measured in millimeters
* Reddened flat areas are neg.

The elderly freq. display false neg. or false positive TB skin test

If positive TB test

* Complete history risk factors
* Symptoms
o Weight loss
o Night sweats
o Hemoptysis
o Fatigue

Early am sputum for AFB

Chest xray


Insert needle through chest wall into pleural space

Aspirate fluid

* Diagnostic
* Therapeutic
* Biopsy

Gastric Secretions

* NG tube


* Culturette/swab
* Wet/dry method
* Nose, throat, wound

Review procedure manual & fill in requisitions.

Nursing Functions for Specimen Collection

* Explain procedure, gain client’s participation
* Collect right amt. of specimen at the right time
* Place specimen in correct container
* Label container accurately

(addressograph), plastic bag

Nursing Functions for specimen collection

* Complete lab. Req.
* Place the specimen in the appropriate place for pick up.
* Document/record specimen sent and anything unusual about the appearance of specimen

Blood glucose levels

* Capillary Puncture
* Reduces Venipunctures
* Clients can perform
* Glucometers
* Chemical reagent strip
* Delegated to those instructed in skill if client’s condition stable

Glucose monitoring

* Ordered ac, pc, hs, fasting, before insulin (sliding scale)
* ? Risks for skin puncture
* Assess area of skin
o Sides of fingers, toes, heels
* Client’s ability
* Normal fasting Bld. Sugar

70-120 mg/100ml

Glucose Monitoring

* Wash hands, glove
* Client wash hands, warm water
* Follow instructions on meter
* Massage /milk finger or puncture site
* Antiseptic swab ( allow to dry completely)
* Wipe away first droplet of blood with tissue/cotton ball

Glucose Monitoring

* Dispose of lancet in sharps container
* Wash hands
* Check puncture site
o Can share reading with client
* Record results
* Proceed as indicated by results

The Value of Measurement

3 benefits to measuring progress and results

* Shows where we are now
* Tells if we are heading toward our goal
* Allows us to make improvements along the way

What we measure gets improved. Peter F. Drucker

* Heightens our awareness
* Helps us focus on what we value and where we are going
* Keeps us on track
* Gives info what is happening along the way and enables us to continue or change depending on desired results

Specimen collection Role of the Nurse.ppt


Laboratory specimen: collection, safe transport and biosafety

Successful laboratory investigations

* advance planning
* collection of appropriate and adequate specimens
* labeling and documentation of laboratory specimen
* storage, packaging and transport to appropriate laboratory
* the ability of the laboratory to accurately perform the diagnostic tests
* biosafety and decontamination procedures to reduce the risk of further spread of the disease
* timely communication of results

Specimen collection:
key issues

* Consider differential diagnoses
* Decide on test(s) to be conducted
* Decide on clinical samples to be

collected to conduct these tests
o consultation between microbiologists, clinicians and epidemiologists

Transport medium

* Allows organisms (pathogens and contaminants) to survive
* Non-nutritive - does not allow organisms to proliferate
* For bacteria – i.e., Cary Blair
* For viruses - virus transport media (VTM)

Some tips

* Laboratory investigation should start as early as possible
* Specimens obtained early, preferably prior to antimicrobial treatment likely to yield the infective pathogen
* Before doing anything, explain the procedure to patient and relatives
* When collecting the specimen, avoid contamination
* Take a sufficient quantity of material
* Follow the appropriate precautions for safety

Blood for smears Collection

Capillary blood from finger prick
+ make smear
+ fix with methanol or other fixative

Handling and transport
Transport slides within 24 hours
Do not refrigerate (can alter cell morphology)

Blood for cultures

Venous blood
+ infants: 0.5 – 2 ml
+ children: 2 – 5 ml
+ adults: 5 – 10 ml

Requires aseptic technique
Collect within 10 minutes of fever
+ if suspect bacterial endocarditis: 3 sets of blood culture

Blood for cultures
Handling and Transport

Collect into bottles with infusion broth
+ change needle to inoculate the broth
Transport upright with cushion
+ prevents hemolysis
Wrap tubes with absorbent cotton
Travel at ambient temperature
Store at 4oC if can’t reach laboratory in 24 hours

Serum Collection
Venous blood in sterile tube
+ let clot for 30 minutes at ambient temperature
+ glass better than plastic
Place at 4-8°C for clot retraction for at least 1-2 hours
Centrifuge at 1 500 RPM for 5-10 min
+ separates serum from the clot
4-8oC if transport lasts less than 10 days
Freeze at -20°C if storage for weeks or months before processing and shipment to reference laboratory
Avoid repeated freeze-thaw cycles
+ destroys IgM
To avoid hemolysis: do not freeze unseparated blood

o Lumbar puncture
o Sterile tubes
o Aseptic conditions
o Trained person

Cerebrospinal fluid (CSF)

Handling and transportation
+ preferably in trans-isolate medium, pre-warmed to 25-37°C before inoculation
+ transport at ambient temperature (relevant pathogens do not survive at low temperatures)

+ transport at 4-8°C (if up to 48hrs or -70°C for longer duration)

Rectal swabs
o convenient
o adapted to small children, debilitated patients and other situations where voided stool sample not feasible

o no macroscopic assessment possible
o less material available
o not recommended for viruses

Stool samples Collection:
Freshly passed stool samples
+ avoid specimens from a bed pan
Use sterile or clean container
+ do not clean with disinfectant
During an outbreak - collect from 10-20 patients

Stool samples for viruses
o within 48 hours of onset
Sample amount
o 5-10 ml fresh stool from patients (and controls)

o fresh stool unmixed with urine in clean, dry and sterile container

o refrigerate at 4°C; do not freeze
o store at -15°C - for Ag detection, polymerase chain reaction (PCR)

o 4°C (do not freeze); dry ice for (Ag detection and PCR)

Stool samples for bacteria
o during active phase
Sample amount and size
o fresh sample and two swabs from patients, controls and carriers (if indicated)

o Cary-Blair medium
o For Ag detection/PCR – no transport medium

o refrigerate at 4°C if testing within 48 hours, -70°C if longer; store at -15°C for Ag detection and PCR

o 4°C (do not freeze); dry ice for Ag, PCR detection

Stool samples for parasites
o as soon as possible after onset

Sample amount and size
o at least 3 x 5-10 ml fresh stool from patients and controls

o mix with 10% formalin or polyvinyl chloride, 3 parts stool to 1 part preservative
o unpreserved samples for Ag detection and PCR

o refrigerate at 4°C; store at -15°C for Ag detection and PCR

o 4°C (do not freeze); dry ice for antigen detection and PCR

Throat swab (posterior pharyngeal swab)
Hold tongue away with tongue depressor
Locate areas of inflammation and exudate in posterior pharynx, tonsillar region of throat behind uvula
Avoid swabbing soft palate; do not touch tongue
Rub area back and forth with cotton or Dacron swab

Nasopharyngeal swab
Tilt head backwards
Insert flexible fine-shafted polyester swab into nostril and back to nasopharynx
Leave in place a few seconds
Withdraw slowly; rotating motion

Nasopharyngeal aspirate
Tilt head slightly backward
Instill 1-1.5 ml of VTM /sterile normal saline into one nostril
Use aspiration mucus trap
Insert silicon catheter in nostril and aspirate the secretion gently by suction in each nostril

Instruct patient to take a deep breath and cough up sputum directly into a wide-mouth sterile container

o avoid saliva or postnasal discharge
o 1 ml minimum volume

Respiratory samples
Handling and Transport
All respiratory specimens except sputum are transported in appropriate media

o bacteria: Amie’s or Stuart’s transport medium
o viruses: viral transport medium (VTM)
Transport as quickly as possible to the laboratory to reduce overgrowth by oral flora

For transit periods up to 24 hours
o ambient temperature for bacteria
o 4-8°C for viruses

Biopsy relevant tissues
+ place in formalin for histopathology
+ place in transport medium for microbiological testing
+ place in sterile saline for isolation of viral pathogens

Post-mortem samples
Post-mortem samples
Handling and transportation
Fixed specimens can be transported at ambient temperatures

+ transport specimens in transport media within 24h at ambient temperature
+ transport specimens in sterile saline at 4-8oC within 48h

Specimen Transport
Storage condition
Purpose/ Lab investigation
Pending test
Throat swab
Isolation, serology
Clotted blood
Whole blood
Virologic Investigations
Storage condition
Purpose/ Lab investigation
Pending test
Throat swab
Amie’s or Stuart’s TM
Isolation Visualization
Bacteriologic Investigations
Water for bacteriology
Chlorinated water - add sodium thiosulphate (0.5ml of 10% solution or a small crystal)
Tap/ pump
+ remove attachments
+ wipe, clean and flame outlet
+ allow to flow (at least one minute)

Water course or reservoir - collect from a depth of at least 20 cm
Dug well - do not allow the bottle to touch the sides of the well

Water for bacteriology Collection
At least 200 ml of water sample from the source
In sterile glass bottles OR autoclavable plastic bottles
+ tight screw capped lid
+ securely fitting stoppers/caps
+ an overhanging rim

Handling and transportation
Test the water sample within 3 hours of collection
+ keep at ambient temperature

If delayed:
+ pack sample on ice
+ test refrigerated sample within 24 hours

Food samples
Collect suspect food earliest
Collect aseptically - sterile tools, containers

Solid Food
o cut 100-200 grams from centre with sterile knife
o raw meat or poultry - refrigerate in a sterile plastic jar

o shake to mix, use sterile tube
o water used for cooking -- 1-5 liters

Contact surfaces (utensils and/or equipment) for food processing
o moisten swab with sterile 0.1% peptone water or buffered distilled water; put the swab in an enrichment broth

Food samples
* Handling and transportation
o As fast as possible
o Keep perishable food at 2-8 °C
o Cool hot food rapidly - put containers under cold running water
o Pack samples to prevent spillage
o Contact the laboratory regarding method of transport and anticipated time of receipt
o Seek help from environmental/veterinary microbiologist

Labeling specimens
* Patient’s name (or Patient Identifier)
* Unique ID number (Research/Outbreak)
* Specimen type
* Date, time and place of collection
* Name/ initials of collector

Patient’s Name/Identifier Unique ID Number
RRR-0023 001712643003
Date, Time, Place of Collection
Specimen Type
Collected by:

Glass slides for microscopy
Label slides individually
o use glass marking pencil
o ensure markings don’t interfere with staining process

Each slide should bear:
o patient name
o unique identification number
o date of collection

Some Tips
* Pre-print labels
* Permanently affix label to the specimen container.
* Glass slides for microscopy labeled individually
* One specimen – one lab request
* Each slide should bear the patient’s name, unique identifier, and date of collection
* Use line listing for multiple patients
* Original documents kept by investigation team

Case investigation form
Epidemiologist sends:
Patient information
o age (or date of birth), sex, complete address

Clinical information
o date of onset of symptoms, clinical and immunization history, risk factors or contact history where relevant, anti-microbial drugs taken prior to specimen collection

Laboratory information
o acute or convalescent specimen
o other specimens from the same patient

Line listing – if large number of patients

Case investigation form
Receiving laboratory records:
Date and time when specimen was received
Name and initials of the person receiving specimen
Record of specimen quality

Criteria for rejecting samples
Mismatch of information on the label and the request
Inappropriate transport temperature
Excessive delay in transportation
Inappropriate transport medium
o specimen received in a fixative
o dry specimen
o sample with questionable relevance

Insufficient quantity

Most of the slides used in this presentation are developed by the Department of Epidemic and Pandemic Alert and Response of the World Health Organization with assistance from:

European Program for Intervention Epidemiology Training
Canadian Field Epidemiology Program
Thailand Ministry of Health
Institut Pasteur


* Communicable Disease Toolkit: Iraq Crisis. Guidelines for the collection of specimens for laboratory testing. WHO, 2003
* Guidelines for the collection of clinical specimens during field investigation of outbreaks, WHO, 2000
* The role of laboratories and blood banks in disaster situations, WHO publication, 2001
* Sampling during avian influenza investigations, 2006
* IDSR guidelines for specimen collection, 2003
* Laboratory Needs for Emergency Situations, 2003
* Overview of Laboratory Structure and Operational Needs for the Iraqi Crisis, 2003
* Costing for sampling materials and diagnostic reagents for the Iraq crisis, 2003

Successful laboratory investigations.ppt


Pre-analytical Laboratory Errors

Pre-analytical Laboratory Errors
By: Tim Guirl MT (ASCP)
Phlebotomy Instructor
North Seattle Community College
Health & Human Services Division


* Identify the significant pre-analytical errors that can occur during blood specimen collection and transport
* Explain the various means of pre-analytical error prevention
* List proactive steps to reduce potential pre-analytical errors associated with blood collection and transport


* Three phases of laboratory testing: pre-analytical, analytical and post-analytical
* Pre-analytical—specimen collection, transport and processing
* Analytical—testing
* Post-analytical—testing results transmission, interpretation, follow-up, retesting.

Phlebotomy Errors

* Phlebotomy is a highly complex skill requiring expert knowledge, dexterity and critical judgment
* It is estimated that one billion venipunctures are performed annually in the U.S.
* Phlebotomy errors may cause harm to patients or result in needlestick injury to the phlebotomist

Pre-analytical errors

* Pre- and post-analytical errors are estimated to constitute 90% of errors
* Errors at any stage of the collection, testing and reporting process can potentially lead to a serious patient misdiagnosis
* Errors during the collection process are not inevitable and can be prevented with a diligent application of quality control, continuing education and effective collection systems

Types of Collection Errors

* Patient Identification
* Phlebotomy Technique
* Test Collection Procedures
* Specimen Transport
* Specimen Processing

Patient Identification Errors

* Errors in correctly identifying the patient are indefensible
* Reasons for patient identification errors
o Proper positive patient identification procedures not followed
+ Patient identification from identification bracelet (inpatients)
+ Patient identification by asking patients to state or spell their full name (inpatients/outpatients)
+ Patient identification by staff or family member if patient unable to identify him/herself

Patient Identification Errors

o Specimen tubes unlabeled
+ Requisition or collection tube labels not affixed to tubes
# Requisition or collection tube labels in bag containing collection tubes
# Requisition or collection tube labels rubber-banded to tubes
# Collection tube labels not affixed to all tubes
# Specimen collection tubes labeled insufficiently with at minimum patient’s full name, date/time of collection, phlebotomist’s initials

Patient Identification Errors

* Collection tubes labeled with the wrong patient
o Wrong computerized labels affixed to collection tubes at bedside
o Collection tubes not labeled at the time of collection
o Collection tubes incorrectly labeled by someone other than the phlebotomist who collects the specimen

Patient Complications

* Some patient variables that affect blood specimens
o Diet
+ Fasting
o Exercise
o Obesity
o Allergies to alcohol or iodine used to clean venipuncture site
+ Use alternative cleanser such as chlorhexidine

Phlebotomy Technique Errors

* Phlebotomy technique is important
o Ensures test result validity
o Minimizes trauma to patient
o Minimizes potential for phlebotomist injury
o Reduces recollections
* Vein selection essential for successful venipuncture
o Three veins in antecubital fossa in order of selection (1) median cubital (2) cephalic (3) basilic

Phlebotomy Technique Errors

* Site Selection
o Avoid sites with IV
+ Use alternative arm or draw below IV to avoid contamination/dilution from IV
+ Document arm if IV
o Mastectomy—avoid site due to lymphostasis
+ Infection risk/alteration in body fluids and blood analytes
o Edematous areas —avoid due to accumulation of body fluids
+ Possible contamination/dilution of specimen

Phlebotomy Technique Errors

o Venous Access Difficulties
+ Obstructed, hardened, scarred veins
+ Veins difficult to locate
+ Use of Alternative sites
# Top of hand/Side of wrist
# Areas to avoid
o Vein Collapse
+ Use of appropriate needle size
+ Smaller evacuated collection tube

Phlebotomy Technique Errors

* Tourniquet Application
o Tourniquet tied too close to the venipuncture site can cause hematoma
o Veins may not become prominent if tourniquet is tied too high (more than 3 to 4 inches above venipuncture site)
o Tourniquet left on longer than one minute can result in hemoconcentration, affecting some test results
+ Tourniquet should be released as soon as needle is in the lumen of the vein and blood flow established

Phlebotomy Technique Errors

* Cleansing of venipuncture site
o Thorough cleaning with alcohol
o Allow alcohol to dry completely to avoid stinging sensation upon needle entry and hemolysis of sample
o Samples such as blood cultures should be collected using iodine to cleanse site to ensure sterility of sample
+ Recollection rate for blood cultures ranges due to contamination is as high as 50% in hospitals with increased costs, patient overtreatment

Phlebotomy Technique Errors

* Correct collection system
o Evacuated tube system (Vacutainer) for large veins in antecubital fossa
o Syringe for small, fragile veins or veins outside antecubital fossa
* Venous access
o Needle entry should be at 15 to 30 degrees depending on depth of vein
o Needle entry should be in same direction as vein, centered over vein
o Anchor vein to prevent movement during needle entry and to reduce pain to patient

Test Collection Errors

* Order of Draw
o Order of draw affects the quality of the sample and can lead to erroneous test results due to contamination with the additive from the previous blood collection tube
* Hemolysis
o Blood collected insufficient to amount of additive in tube,
o Traumatic venipuncture
o Blood collected from area with hematoma
o Vigorous shaking of tubes after collection
o Milking the site when collecting capillary samples and blood collected using a small diameter needle.

Test Collection Errors

* Timing of Collection
o Timed Draws
o Therapeutic Drug Monitoring
+ Peak and trough collection times
o Basal State Collections
+ Fasting requirements—no food or liquid except water
o Specimens affected by time of day, for example, cortisol

Test Collection Errors

* Improper collection tube drawn for test ordered
* Collection tube not completely filled
o Example—light blue top tube for Coagulation Studies. Incomplete filling results in specimen dilution and erroneous Prothrombin and aPTT test results.

Test Collection Errors

* Capillary Collections—finger stick or heel stick
o Appropriate site
+ Heel stick—sides of the bottom surface of the heel
+ Finger stick—third or fourth fingers, perpendicular to fingerprint lines on fleshy pads on finger surface
o Warming—Warm before collection to increase capillary blood flow near skin surface
o Cleaning—cleanse site with alcohol and allow to air dry

Capillary Collections

o Massaging site to increase blood flow
+ Milking site can cause hemolysis or tissue fluid contamination
+ Finger sticks—roll fingers toward fingertip at 1st finger joint several times
+ Heel sticks—gently squeeze infant’s heel before performing puncture.
o Perform puncture while firmly squeezing finger or heel
o Wipe away first two drops of blood
+ Ensure that full blood drop wells up each time

Capillary Collections

o Avoid touching capillary collection tube or micro collection tube to skin or scraping skin surface
+ Contaminates puncture site
+ Blood may become hemolyzed
o Mixing micro collection tubes with additive frequently to avoid micro clots
o Collecting tubes with additives first
o Protecting tubes for bilirubin from light

Blood Specimen Transport Errors

* Transport of blood specimens in the proper manner after collection ensures the quality of the sample
* Timing
o Some specimens must be transported immediately after collection, for example Arterial Blood Gases.
o Specimens for serum or plasma chemistry testing should be centrifuged and separated within two hours

Transport Errors

* Temperature
o Specimens must be transported at the appropriate temperature for the required test
+ On ice—ABGs, Ammonia
+ Warmed --98.6 degrees (37 C), cryoglobulins
+ Avoid temperature extremes if transported from via vehicle from other collection site
* Transport Container
o Some samples need to be protected from light, for example, bilirubin
o Transport in leak-proof plastic bags in lockable rigid containers

Error Prevention

* Phlebotomy Education
o Phlebotomists should have completed a standard academic course in phlebotomy and undergo thorough on-the-job training under the supervision of a senior phlebotomist
* Continuing Education
o Phlebotomists should participate in regular educational competency assessments (written and observational)
o Professional Licensure
* Phlebotomy Staffing
o Adequate staffing to maintain collection standards
* Technology
o Use of barcode scanners for patient identification

Questions and Discussion

* How are pre-analytical errors prevented in your laboratory?
* What technology do you use to prevent human error?
* What systems does your hospital use to prevent errors by non-laboratory staff collecting blood?
* What pro-active improvements would reduce the number of pre-analytical errors?

Pre-analytical Laboratory Errors.ppt

All links posted here are collected from various websites. No video or powerpoint files are uploaded on this blog. If you are the original author and do not wish to display your content on this blog please Email me anandkumarreddy at gmail dot com I will remove it. The contents of this blog are meant for educational purpose and not for commercial use. If you use any content give due credit to the original author.

This site uses cookies from Google to deliver its services, to personalise ads and to analyse traffic. Information about your use of this site is shared with Google. By using this site, you agree to its use of cookies.

  © Blogger templates Newspaper III by Ourblogtemplates.com 2008

Back to TOP