1. Labs – What does that really mean?
23rd Annual AONP Conference, MidWest City, Oklahoma
10/18/17, 4:30pm
Jennifer Jameson, MT (ASCP) & Rochelle Colby (MLS)CM

2. Objectives Introduction Laboratorians Specimen Collection
General Laboratory (Blood Bank, Chemistries, Coag, Blood Gas, Point of Care (POC))
Urinalysis
Microbiology
Hematology
Pathology

3. Presenters Jennifer Jameson MT(ASCP) Rochelle Colby MLS (ASCP)CM
25 years of clinical experience
23 years in Microbiology
20 years at Cancer Treatment Centers of America
7 years of clinical experience
3 years as Lead Hematology Tech
6 years at Cancer Treatment Centers of America

4. Who are we? Laboratorians – MT/MLS/CLS/MLT
Bachelor degree (MT/MLS/CLS) or associates (MLT)
Medical Technologist, Medical Laboratory Scientist, Clinical Laboratory Scientist
Medical laboratory scientist degree (Bachelor) programs are set up in a few different ways.
In 3+1 programs, the student attends classroom courses for three years and complete a clinical rotation their final year of study.
In 2+2 programs, students have already completed their lower division coursework and return to complete their last two years of study in a CLS program.
In 4+1 program, students who have already completed an undergraduate program return to complete a year of medical laboratory training. The training is typically completed at a clinical site rather than a college.
The core curriculum in medical technology generally comprises 20 credits in clinical chemistry, 20 credits in hematology, and 20 credits in clinical microbiology.
Clinical Rotation
Student experiences hands-on learning in each discipline of the laboratory and performs diagnostic testing in a functioning laboratory under supervision. With limited or no compensation, a student in the clinical phase of training usually works 40 hours per week for 20 to 52 weeks.
National registry certification exam
Administratively independent of the ASCP, the Board of Certification (BOC) has certified more than 450,000 people since it was founded in 1928 and represents the gold standard for certification of pathologists’ assistants and laboratory professionals
The American Society for Clinical Pathology (ASCP) is the world’s largest professional membership organization for pathologists and laboratory professionals. Our mission is to provide excellence in education, certification and advocacy on behalf of patients, pathologists and laboratory professionals across the globe. With more than 100,000 members, the society’s influence has guided the application and evolution of the pathology and laboratory medicine specialty since 1922.
Medical Laboratory Scientists who are certified and in good standing with the approved National Medical Laboratory Science Council or the American Society for Clinical Pathology (ASCP) are entitled to use the credential "MLS" after their names (after Oct. 2009). They are entitled to use the title "Scientist." Formerly before the merger between ASCP and the National Credentialing Agency for Laboratory Personnel (NCA), medical laboratory scientists certified by (ASCP) were entitled to use the credential "MT" (for medical technologist) and if credentialed by (NCA), the credential "CLS" (Clinical Laboratory Scientist) was used.
The Credential Maintenance Program is required for all individuals who are ASCP certified beginning January 1, 2004 in the following categories and for all active NCA certificants who are not ASCP certified prior to January 1, Medical Laboratory Scientist (MLS), Medical Laboratory Technician (MLT)
For generalists (MLS/MT & MLT) Minimum of 36 points (hours) to include: 1 point minimum in safety, 2 points minimum required in each domain (blood banking, chemistry, hematology, microbiology), and remaining points in area(s) of laboratory specialty, immunology, molecular diagnostics, management, education, and/or other related laboratory areas of interest.

5. How do labs affect your diagnosis and treatment decisions?
It is estimated that 70% of all health care decisions affecting diagnosis or treatment involve a pathology investigation. Decisions on an individual’s diagnosis, treatment and subsequent therapeutic monitoring are often dependent on a range of pathology-based results.
Lab and Pathology Results affect:
Imaging/Radiation
Pharmacy
Surgery
Nutrition
Oncology
Clinical Care
Source Clin Biochem Rev Aug; 34(2): 43–46. :

6. Ordering Lab Tests
Order lab tests relative to patients clinical symptoms
use to Rule out/Rule in
Correlate lab results with the clinical impression
Use Positive Predictive Value
the probability that subjects with a positive screening test truly have the disease
Probability of False Positives - look at the whole picture
We use these terms when evaluating laboratory tests before implementing them.

7. Results are ONLY as good as the specimens that we get
Lab Mantra
Results are ONLY as good as the specimens that we get
Patient identification-- two identifiers
Source on specimen--write it on there and be specific
Date, time and collector’s initials -- part of good technique
Transports/tube types
tests can be temperature dependent
some tests cannot have light exposure
others need to be in preservative within minutes
Examples of what happens with poor collections
Redraws happen sometimes-- we want to make sure
Examples of poor collections: clot taken out of a CBC tube (critical platelets) and critical high potassium on chem due to drawn in purple top (contains K) and then poured into a green top

8. Just a meme to hit home how important it is to label the actual specimen :)

9. And another meme to hit home that the lab personnel don’t know room numbers but they do know names :) and MR numbers :) and birthdates :)

10. Specimen Collection Importance of: Order of draw Transport media
Timing
Orders
Order of draw – if not properly – cause contamination – false results – leads to improper treatment
Transport media – microbiology – integrity of specimen
Timing – troughs, peaks, specific testing requirements…
Orders (placed) – by law cannot do testing without an order – The longer a specimen sits the higher chance of wrong/false results
Lab calls for recollects when notice variance in results – wanting to verify it was correct before patient is treated is with those results

11. General Lab - Chemistry
**Methodologies are important**
not all labs/tests are interchangeable for result comparison.
Chemistries: Methodologies
Photometry- measurement of light by photodetector (absorbance, turbidity, fluorescence)
Potentiometry - electrical potential between 2 electrodes - Na, K, Cl testing
End Point Reactions - chemical reaction
Immunoassay (antigen/antibody reactions) - Drug levels (Dig, Vanc), Hormone (TSH, Estrogen), Tumor Markers (PSA, AFP, CA-125)
ELISA, Western Blot, FISH, PCR
Mar 2017

12. General Lab - Blood Bank
Patient ID #1 (blood bank bracelets, scanning ID system)
Most specimens only good for 72 hours
Make sure to answer questions (past transfusions, pregnancy, transplants)

13. General Lab - Blood Gases
sensitive specimens

14. General Lab - Point of Care

15. General Lab - Coagulation

16. Urinalysis 3 parts to UA Physical Examination (Color, Clarity, etc)
Biochemical Examination (Blood, Leukocytes, Glucose, Protein etc.)
Microscopic examination (WBC, RBC, Bacteria, etc.)

18. Urinalysis Collections – Clean catch, cath, neph., suprapubic tap
Contamination/obstructive analysis – not properly collected/clean catch, sitting prior to analysis, drugs (pyridium)
Differences between Macroscopic (dipstick) & Microscopic findings
Notes on LeukEst and Hemoglobin: cells may not be present in the microscopic and have high LE/HgB…. it’s measuring what’s inside the cell… recollect and repeat on fresh urine
Just because a urine is Nitrite negative doesn’t mean it’s negative for UTI. Not all bacteria produce Nitrite (like Enterococcus and Staphylococcus) or viral infections

19. Microbiology Step by Step overview MICs and more Blood Cultures
Bacterial vs Viral
Antibiotic Stewardship
New tests/methodologies

20. Microbiology

21. Microbiology Setup
First, the fabulously perfectly labeled and tightly sealed specimen container arrives in the lab
Then identification and orders are reconciled and specimen is received
It is taken into microbiology where it is “planted” or “plated” onto agar media
At CTCA, all specimens are plated in the BSC (biological safety cabinet) a.k.a The Hood (tell Brucella story)

22. Microbiology Setup
Incubate for 24 hours….. and you get…..

23. Microbiology Setup
Using a sterile, calibrated wand, a few colonies are picked from the plate
Wand is then placed into a specific amount of fluid to make the organism dilution work in the antibiotic panel
The dilution is poured into a tray

24. Microbiology Setup
A 96 well pipettor is used to draw up the fluid from the tray
And then it is released at once into the antibiotic panel
And it is incubated another 24 hours

25. Here is the final product :)
Top part with pretty colors is the identification portion of the panel. Each well corresponds with a number and the number adds up to a biotype.
Biotypes are connected to an organism name, in this case, this is E coli
The bottom of the panel is the susceptibility portion of the panel. You can see the wells are usually in doubling dilutions, with the drug getting stronger as it goes to the right. This one is resistant to Ampicillin, Pipercillin, Amp/sulbactam, Cephalothin, and to the fluroquinolone class of drugs, and it is intermediate to Augmentin.
Where the arrow is pointing, is called an MIC. For instance, Gentamicin… <=4 is susceptible…. equal to 8 is intermediate…. >16 is resistant.. this panel has 2 wells so if it grew in the 4 well, the MIC would be 8, which is intermediate. If it grew in both the 4 and the 8 wells, it is greater than 8 which means resistant (even though there isn’t a 16 well to say “resistant”, anything larger than 8 would be resistant)

26. Microbiology Susceptibilities
MIC = Minimal Inhibitory Concentration
The lowest antimicrobial concentration that completely inhibits visible bacterial growth (this means the next well could have some tiny bit of growth).
Given in units of mcg/ml. The amount of active drug in micrograms per amount of broth in milliliters (how the wand, bottle and panel all work together).
This number is useful when the interpretation is Intermediate. If Susceptible, the drug can be used. If Resistant, it shouldn’t be used. But Intermediate means if you can get the drug in a high enough concentration into the patient’s system, you may be able to use the drug.
But it isn’t always safe to do this so consult with an ID expert or a Pharm D first (there’s a whole other level of PK/PD and how drug diffuses throughout the body… and a whole other lecture needed for that)
Watching the MIC levels over time help us monitor resistance
MIC is a type of susceptibility. You will see numbers like <=2 next to an interpretation of susceptible-intermediate-resistant, or S-I-R.

27. Microbiology Susceptibilities
MIC cont’d
Why do the MIC numbers vary? Depends on the bug AND the drug!!
Example: A MIC of 8 for vancomycin versus Enterococcus is Intermediate. A MIC of <=32 for nitrofurantoin (Furadantin) versus Enterococcus is Susceptible.
The MIC breakpoints (i.e. cut off values for S-I-R) are different for all the different classes of antibiotic. They are not comparable or interchangeable. Vancomycin is a glycopeptide and nitrofurantoin is in the class Nitrofurans.
Example: To detect significant resistance to cefepime in Streptococcus
pneumoniae, the dilution scheme uses a max concentration of 4 mcg/ml;
however, to detect cefepime resistance in E coli, the required max
concentration is 16 mcg/ml.
Concentration of a drug level to detect resistance is different in each organism.

28. Microbiology Susceptibilities
Kirby Bauer and Etest methods
Kirby Bauer = lawn of growth of your bug and drop antibiotic disks on… incubate and read out in millimeters (get S-I-R interpretations only)
ETest = combination of KB and MIC… make a dilution of organism, make a lawn of growth and add an antibiotic strip that has gradual increase of drug and can read
like an MIC (best of both worlds)

29. Microbiology Blood Cultures
Typically held 5 days total before final
Contamination rates ideally should be at or below 3% (per CLSI guidelines)
A contaminated culture costs both the hospital and the patient:
with longer in-house stays
greater antibiotic use
increased diagnostic testing
Should be calculated monthly to monitor the rate and to help educate those that draw them
If you have a positive culture at 36 hours old, one bottle of 4, and gram stain shows Gram Positive Cocci…. true positive? contaminant? Clinical symptoms and port vs peripheral draw… tricky!!!
CTCA’s number one pathogen…. coagulase negative Staphylococcus (we have a lot of VAD’s and that means biofilm and that means colonization can turn to pathogen with immunosuppression)
And don’t forget… fungus!! Yeast likes to live in plastic lines!!
CLSI = Clinical and Laboratory Standards Institute. This is a committee of really smart people that have a lot of time to research stuff and set the standards in the lab for testing methodologies to writing
procedures to setting breakpoints and benchmarks. They are our gurus :)

30. Microbiology Bacterial vs Viral
Flu season is coming up!!
New guidelines coming out
(compliance by Jan 2018)
Culturing for viruses is almost
obsolete.
Best method is molecular (viruses
are all DNA or RNA so easy to
replicate them
We’ve seen viral throat infection
that mimicked strep--beware!!

31. Microbiology Antibiotic Stewardship
Here’s some scary statistics about microbiology and antibiotic usage:
An estimated 50% of antibiotic use in humans is unnecessary and inappropriate.
Antibiotic-resistant bacteria cause over 2 million illnesses and 23,000 deaths annually in the US (as of 8/14/17).
Antimicrobial resistance is an increasingly serious threat to health at the patient, community and global level.
Prescribing of second-and third-choice drugs due to resistance of first-choice drugs resulting in less effective, more toxic and more expensive drugs being used.
“At current rates of antimicrobial resistance, we estimate that 2.5 million people will be lost to drug-resistant ‘superbugs’ in 2020; 5.9 million will be lost in 2030; 15 million will be lost in 2050”.
The rate of resistance is increasing faster than new antimicrobials are being discovered (Methicillin introduced in 1960, resistance detected in 1962; Levofloxacin introduced in 1996, resistance detected in 1996).
Source: and

32. Microbiology Antibiotic Stewardship
This topic was important enough to be mentioned in the 2014 Presidential State of the Union address.
By March of 2015, a National Action Plan, that charged key federal departments and agencies, was created to combat the rise of antibiotic-resistant bacteria.
Most hospitals, by now, have implemented an Antibiotic Stewardship Program (ASP). This is usually led by Pharmacy and includes clinicians, Quality, Infection Prevention and Laboratory departments.
What can you do to help?
Get involved with your ASP and get educated (and educate your patients, too)
Check culture results to make sure bug-drug combo is working
D/C drugs that aren’t needed
Ask to see your lab’s antibiogram (a chart that summarizes the percent of pathogens susceptible to different antibiotics; done retrospectively)

33. Microbiology Newer Methodologies
Stool lactoferrin -- replaces stool for polys
Cryptococcal Antigen -- replaces India Ink test
EIA/ELISA methods-- for common parasites
Chromogenic agar -- selective for certain organisms and will turn color when that organism grows
PCR/molecular -- best way to detect viruses, toxins, harder to grow organisms
C difficile (need to be doing a 2-step algorithm--best practice)
Respiratory pathogens
Stool pathogens
CSF pathogens
MALDI TOF -- Matrix-Assisted Laser Desorption/Ionization Time Of Flight-- basically a mass spectrometer with pulsing lasers and ion mirrors and electric fields that give you an organism identification (no susceptibility yet) in about 10 minutes!! The more that are put into use, the larger the database grows and the more reliable it becomes. In use around the country especially being placed into Health Depts
Check sensitivity and specificity before implementing a new system-- may not be the best choice for what your facility needs
With microbiology taking so long, it is a challenge to find testing methods that will decrease the time for results (48+ hours for susceptibility results from time of culture set up)
Some cultures have too much going on like stool… PCR is a great way to find the needle in the haystack
MALDI’s are being used for fungus and AFB organisms… sometimes just too dangerous to grow an organism

34. Hematology

35. Hematology Top vs. Bottom instrument vs. manual diff
indications for manual
Flow Cytometry - technology that is used to analyze the physical and chemical characteristics of particles in a fluid as it passes through at least one laser. Cell components are fluorescently labelled and then excited by the laser to emit light at varying wavelengths
Top (Hemogram)
Bottom (Differential)
Use of flow cytometry would allow evaluation of many more cells than possible by morphology, allowing for more accurate enumeration of populations present at low numbers. Additionally, flow cytometry allows for specific positive identification of cell types, thereby decreasing misclassification of cells with unusual morphology
Suspect Flags (Manual Differential)

36. Flow Cytometry - Differential Scattergram

38. Suspect flags from flow cytometry = manual differential review/differential

39. Possible Peripheral smear from previous scattergram
Figure 1. Chronic phase of chronic myelogenous leukemia. The peripheral blood smear is characterized by an elevated white blood cell count showing a left shift in granulocyte maturation, with increased myelocytes. Eosinophilia and basophilia are characteristic (Wright-Giemsa stain, X 100)
Source:

40. Hematology
Left shift – particular population is “shifted” towards more immature precursors (ie bands, metamyelocytes, myelocytes etc).
Right Shift - Neutrophil count will be higher than normal range (predominant cell w/higher than normal absolute value) without the presence of immature cells (bands, metas, myelos)
Term Left Shift came from 1920s publication of Josef Arneth, in which neutrophil maturity was correlated with segment count. A graphical representation was made and the fewer the segments, the further left was median, hence left shift. Now used for indication of infection.
Right shift - giant neutrophils generally not noted on differential but called as regular neutrophils
Source:

41. Hematology
Toxic Changes: Matters for distinction of infection especially in leukopenic patients.
Will be noted on the manual differential
Dohle Bodies
Toxic Granulation
Vacuoles

42. Pathology Specimens in Pathology flow overview
Specimens received in the pathology lab
Specimen Macroscopically Examined
"grossing" is the process by which pathology specimens are inspected with the bare eye to obtain diagnostic information, while being processed for further microscopic examination. Gather diagnostically critical information in this stage of processing, including the stage and margin status of surgically removed tumors
Specimen is then sectioned and processed (overnight process)
Once processed it is then thinly cut and placed on slide for microscopic evaluation
May need special stains
identify organisms/diagnosis (add 1-2 days)
May be sent out for additional testing (add 1-2 days)
If Cancer and needs molecular testing it is sent out (add 1-2 weeks)
**Note: Benign FNA or Biopsy does not rule out Cancer
Examples of why: May have missed the spot when performing procedure, QNS sample for adequate processing, overwhelmed with inflammation so unable to accurately diagnose. Use Positive Predictive Value and clinical picture to decide if to re-biopsy

43. Questions?