Increase the value of your diagnostics and your value as a diagnostician - Dr. Marie Culhane, Associate Clinical Professor, Veterinary Population Medicine, College of Veterinary Medicine, University of Minnesota, from the 2013 Allen D. Leman Swine Conference, September 14-17, 2013, St. Paul, Minnesota, USA. More presentations at http://www.swinecast.com/2013-leman-swine-conference-material

1. Increase the Value of Your
Diagnostics and Your Value as a
Diagnostician
Marie Culhane, DVM, PhD,
Jim Collins, DVM, PhD, Dipl. ACVP,
Rebecca Davies, PhD
University of Minnesota
Veterinary Diagnostic Laboratory
St. Paul, MN

2. $ for Dx
• All veterinary expenses under scrutiny.
– Properly invest time and effort into all
decisions
• Medications
• Vaccinations
• Pig movements
• Diagnostic submissions
– But what if the results you receive from the
diagnostic laboratory just do not make sense?

3. Presentation Overview
and Steps to Improve your Diagnostic Value
• Proper sampling technique
• Submission of samples
• Interpretation of results

4. Keys to Diagnostic Success
• Goal:
– The Accurate and timely diagnosis of disease
• Decrease losses
• Facilitate therapy
• Protect the customer
• Specimen selection and quality can impact the
diagnostician’s ability to accurately find the
cause of death or disease

5. How do you get there?
Do a proper disease investigation!

6. Starting a Disease Investigation
• What Problem are you Investigating?
– Clinical signs?
– Unexpected/undesired serology results or
slaughter check findings
– Production changes

7. Disease Investigation
Preparation
Pig
selection
Time
Thermometer
Clinical
Observation
Euthanized
Treated
vs. dead
vs. Untreated

8. Disease Investigation
• Necropsy
– Tools
– Assistants
– Euthanasia method
– Environment
– Begin

10. General Tissue Sets
• Respiratory
– Fixed lung, tonsil, turbinate, trachea, heart, liver,
spleen, kidney, lymph node
– Unfixed lung, snout, tonsil, liver, spleen, kidney,
lymph node
– Serum

11. Quantity of specimens
• Unfixed (“fresh”) tissues
– Generous sample
• Approximately 3 X 10 cm (hockey puck)
– If lung, airways
– 12 to 15 cm length of intestine
– Lesions
• Multiple, segmental

14. Quantity of Sample
• Fixed tissues
– Less than 1 cm thick, less than 5 cm long
– Lesions – multiple, segmental
– Intestines unopened
• Contents
– Brain half (as is)
– Preserved in 10% buffered formalin
– Formalin to tissue ratio = 10:1

17. Specimen Submission
• Refrigerate unfixed tissues, do not freeze
• Frozen gel packs = weight of tissue
• Whirl-pak bags
– Suitable for unfixed tissues
• Sealed containers
– Better for formalin fixed tissues

18. Cecal
contents

19. Swabs
• Essential diagnostic tool
– Allows you to transport fastidious organisms to
the lab to increase chance of detection
– Allows you to access sites that may be difficult to
open (e.g. brain through foramen magnum)
– Allows you to sample quickly and with decreased
contamination from knife or hands

20. Swabs
• Appropriate swabs to use are synthetic (Dacron
or Rayon) on plastic shafts.
• The type of media can vary (most commonly used
are liquid Stuarts and Hank's media), but be
careful to avoid any agar gel-type media.
– In human microbiology laboratories, inhibition of PCR
by agar from transport media and from calciumalginate-tipped swabs have been reported.
• One inexpensive, multi-purpose swab is BBL
CultureSwab Liquid Stuart, Single Swab (pkg of
50), Product Number 220099.

22. Shipping formalin-fixed tissues
• All Whirl-Pak bags will leak eventually
– Increased leaked with improper closure or if
overfilled.
• Leaking formalin kills bacteria culture negative
• Leaking formalin kills viruses  culture negative.
– PCR may still work on the above if not overly exposed
• Leaking formalin will cause a package to be
discarded by courier as “Hazardous Waste”

23. Specimen Submission
• Outer bag, labeled
– Protect shipping container
• Submission form
– Outside of cooler, in cardboard box
• Same day or overnight delivery

27. Sample handling still counts and
still costs!
Rebecca L Davies, PhD
Quality Assurance Coordinator
Veterinary Diagnostic Laboratory
University of Minnesota

29. We want to get it right the first time.
High degree of accuracy =
minimal number of errors

30. Errors in laboratory medicine
The interval between the time a sample is
collected until the result is reported to the
veterinarian can be divided into 3 phases:
– Pre-analytical phase
– Analytical phase
– Post-analytical phase

31. Errors in Laboratory Medicine
Categories of error
– Pre-analytical phase(Farm / Clinic)
– Analytical phase
(Laboratory)
– Post-analytical phase
(Shared responsibility)

32. Pre-analytical phase errors
•
•
•
•
•
Animal status and information sharing
Specimen collection
Specimen processing
Specimen transport
Specimen delivery to instrument for analysis

33. Analytical phase errors
All the steps involved in the actual analysis of
the sample in the laboratory
– “lab error”: mis-pipetting, fibrin interference,
equipment errors, tube order errors, transcription
errors
– Lack of sufficient QA/QC
– Assay interferences
– Lack of validated assays
– Inappropriate method choice

34. Post-analytical phase errors
•
•
•
•
Reviewing results for discordance
Interpretation and utilization of results
Appropriateness of reference intervals
Post analysis sample storage

35. Errors in Laboratory Medicine
Categories of error
– Pre-analytical
(Clinic + Laboratory)
– Analytical
(Laboratory)
– Post-Analytical (Clinic + Laboratory)

36. Pre-analytical procedures:
Sample handling hoops
Sample labeling
Tube type
Tube fill level
Storage time/temp
Transport time/temp
Condition received at lab

37. Animal Status and Information
Sharing Errors: Clinical Examples
• Reason for Submission
– Diarrhea
• Age of Pig
–3
• Sample
– Feces

38. No Age Unit Listed
• 3 days, 3 weeks, 3 months, 3 years?
• 3 days old pig
– rotavirus, TGEV, Salmonella sp., E. coli, Clostridium
perfringens, and Clostridium difficile
• 3 weeks old pig
– rotavirus, TGEV, aerobic bacteria, and protozoan parasites.
• 3 years old pig
– TGEV, Salmonella sp., Brachyspira sp., Lawsonia
intracellularis, Yersinia enterocolitica, and metazoan
parasites.

39. Specimen Collection Errors:
Clinical Examples
• Reason for Submission
– Pale pigs
• Age of Pig
– 2 wks
• Sample
– ???

40. Mycoplasma haemosuis infections and
anemia in piglets
• Whole blood in EDTA submitted
• (purple top tube)
– M. haemosuis detection by PCR
• Serum submitted
• (red top or serum separator tube)
– M.haemosuis antibody detection
– PCR test cannot be performed
– Serology test for M. haemosuis antibodies in piglets is not
interpretable
• May represent maternal antibodies
• May be false negative as antibody production to M. haemosuis
occurs in waves

41. Sample Handling and Specimen Processing
Errors: Clinical Examples
• Centrifugation at high speeds, long times, high temp
– destroy viral RNA making it undetectable by PCR
• Accidental freezing of formalin-fixed tissues 
freeze-thaw artifacts
– destroy tissue integrity
• Improper insulation and refrigeration of tissues or
serum  high temperatures  destroy pathogen
RNA or DNA or promote decomposition.

42. Stability of PRRSV in Laboratory Settings
Stability of PRRS in Serum
16
14
vRNA Qty.
12
10
25 degrees
8
4 degrees
6
4
2
cutoff
Cut off = 0.789
0
0
20
40
60
80
100
120
140
Tim e (hours)
Minnesota Veterinary Diagnostic
Laboratory
160
180

43. Stability of Flu RNA in media
37 C
25 C
4C
Qty of vRNA
20
15
10
5
0
0
24
48
72
Time (hrs)
96
120

44. Analytical phase errors
All the steps involved in the actual analysis of
the sample in the laboratory
– “lab error”: mis-pipetting, fibrin interference,
equipment errors, tube order errors, transcription
errors
– Lack of sufficient QA/QC
– Assay interferences
– Lack of validated assays
– Inappropriate method choice

45. Post-analytical Phase
•
•
•
•
Post-analysis storage
Reviewing results for discordance
Interpretation and utilization of results
Appropriateness of reference intervals
– Liver iron levels in pigs very greatly by age and by
supplementation or diet status.

46. Which errors occur with the
greatest frequency?

47. A look at the literature
Errors in Laboratory Medicine: Bonini P, et al.
Clin Chem 48:5, 2002.
Evaluated 8 years of literature (Medline and
hand searches) based on searches for “mistakes,
blunders, errors, problem or defect associated
with laboratory or medical laboratory”.

48. A look at the literature
Errors in Laboratory Medicine: Bonini P, et al. Clin
Chem 48:5, 2002.
“even when different study designs, patient numbers,
and discovery techniques were used, the distribution of
errors across the different phases of the entire testing
process was very similar”
“ In particular, all available studies demonstrated that a
large percentage of laboratory errors occur in the preand post-analytical phases, with fewer mistakes (13-32%)
occurring during the analytical phase”

49. A look at the literature
Mistakes in a stat laboratory: Types and
frequency. Plebani M et al. Clin Chem. 43, 1997.
Analyzed 40,490 stat results
Found 189 errors (0.47%)
68% were pre-analytical
13% were analytical
18% were post-analytical

50. Reducing Laboratory Errors
A Shared Responsibility

51. Veterinarians Should Be Able To:
– carefully observe and describe diseases;
– correlate hx, physical exam, clin signs, and production
data;
– know when, how, what type and to what extent of lab
investigations to undertake;
– understand and interpret the significance of the
laboratory results obtained;
– use knowledge of pathological principles to formulate
possible diagnoses;
– develop hypotheses about underlying mechanisms,
– explain the basic mechanisms underlying the dz

52. Examples of Proper Disease
Investigations
• Clinical Signs: Increased Coughing
• Plan: Visit Farm, Collect Samples, Submit to
Lab, Get Results, Make Decision
– Timeliness of the decision = URGENT
• Movement to another farm as soon as possible
• Options for shipment:
– Drive to nearest lab
– Morning delivery by courier (FedEx AM)
• ALERT THE LAB

55. Example of Proper Disease
Investigation
• Prioritize your differentials and/or work with
the laboratory to decide which tests to do first

56. Prioritize your Differentials
• Which diseases are most important to rule out
or rule in
• Which tests can be done the most rapidly

57. Which Tests Can be Done Rapidly?
• Antigen Detection Tests
– Antigen Capture
• 15 minutes, but decreased sensitivity
– PCR
• Real-time approximately 4 hours but specimen type
affects processing time
– Virus isolation
• Requires viable virus and proper cell typedays

58. Which Tests Can Be Done Rapidly?
• Serology Tests
– ELISA
• 2 to 4 hours
• Detects antibodies so exposure to pathogen of concern
must have already occurred
– SN or IFA or HI
• 12 hours or more because virus and serum interactions
must occur
• Detects only strain specific antibodies

59. Proper Disease Investigation
• Respiratory disease in replacement gilts at
farm of origin
• Lab alerted to “RUSH” Results upon receipt
• Prioritized Disease Differentials
– PRRSV, Mycoplasma hyopneumoniae, and
influenza A virus

60. Clinical Signs: Coughing and nasal discharge. 5 % of the pigs are
sick from a herd of 1,800.
Specimens:
Five porcine nasal swabs with IDs as follows:
– 1 = A(temp 105.3)
– 2 = B(temp 104.9)
– 3 = 300c
– 4 = 606d
– 5 = 119841H
Two sets of fixed and unfixed pig tissues with IDs as follows:
– 6 = U4459(coughing)
– 7 = Q448 (no clinical signs)

61. • Gross Exam: No gross lesions were reported by the
submitter.
• Histopathology:
– Pig 6 = U4459 (coughing) - Lungs - characteristic lesions of
Mycoplasma hyopneumoniae infection consisting of
marked lymphoplasmacytic peribronchitis and
perivasculitis with moderate edematous and purulent
bronchopneumonia and occasional bronchiolitis. Liver,
heart and spleen - no lesions.
– Pig 7 = Q448 (no clinical signs) - Lung, heart, liver, and
spleen - no lesions.

62. Diagnostic Results
•
•
•
•
•
•
•
•
1 = A(temp 105.3), positive for Mycoplasma hyopneumoniae via nasal swab PCR,
negative for influenza A virus vial nasal swab PCR.
2 = B(temp 104.9), positive for Mycoplasma hyopneumoniae via nasal swab PCR,
negative for influenza A virus vial nasal swab PCR.
3 = 300c, negative for Mycoplasma hyopneumoniae via nasal swab PCR, negative
for influenza A virus vial nasal swab PCR.
4 = 606d, negative for Mycoplasma hyopneumoniae via nasal swab PCR, negative
for influenza A virus vial nasal swab PCR.
5 = 119841H, negative for Mycoplasma hyopneumoniae via nasal swab PCR,
negative for influenza A virus vial nasal swab PCR.
6 = U4459(coughing), marked lymphoplasmacytic peribronchitis and perivasculitis
with moderate edematous and purulent bronchopneumonia, M. hyopnepumoniae,
P. multocida.
6 = U4459(coughing), PCV2 positive PCR on tissue homogenate (no lesions of
PCVAD in tissues examined).
7 = Q448(no cs), PCV2 positive PCR on tissue homogenate (no lesions of PCVAD in
tissues examined).

63. Is this a complete and proper
investigation? YES
• Was it PRRS? NO
• Was it Flu? NO
• Was is Mycoplasma? YES
– How do you know with any confidence?
• Histo, lesions, clinical signs, and detection all together
confirm the diagnosis.

64. An Example of an Incomplete
(albeit still useful and diagnostic)
Disease Investigation Example

65. A Case Study
Can you DX resp dz without
necropsy???

66. -----Original Message----From: Bill
Sent: Thursday, June 03
4:35 PM
To: Marie
Subject: Submission to arrive tommorrow
Dr. Culhane,
I have sent a case attn to you concerning a respiratory outbreak in a group of 350 gilts that
were introduced into a sow unit 3 weeks ago. (There is no history in the package, so here
goes.)
This group of gilts has 50% poor performing gilts at this time however 4 weeks ago when I
tested in the I/A the performance was good, even above expectations considering that these
gilts were inoculated 8 weeks previously with live PRRS serum. At the 8 week post entry, the
testing was positive ELISA for PRRS and negative PCR PRRS so they were allowed to enter the sow
unit. While in I/A, the gilts also received Pfizer's FarrowSure PLE-SIV h1n1 and h3n2. This
has been standard for 2 years. Three weeks post entry a cough started. Three died this week
but not able to necropsy any of these. As said above, 50% are showing low feed intake, 20%
hard breathing, 3% looking chronic respiratory disease, but none today looking like death is
likely. Temps were variable on those coughing or hard breathing with most at/less than 104,
2/16 above 104. The cough is not outstanding in nature, more dry with only a few deep. There
is no current involvement in the just previously introduced group of gilts or any suspicions
in the rest of the sow herd. My primary concerns are an unprotected SIV strain, mycoplasma
since this source of gilts may have been negative, and a possibility of ascarids complicating
the situation. Fecals are in progress. I took 16 nasal swabs and sera for an initial
exploration. Necropsy was not done today but planned if any deads. I would like to use the
nasal swabs for SIV PCR, possibly mycoplasma PCR, and others pending results. The serum will
be ELISA PRRS positive but is there value in PCR for PRRS for possible exposure to an
alternative strain? Serology for SIV would have value? I have retained a second set for later
use so acute/convelescent testing can also be done in 2-3 weeks. I hope you can give me some
help.
Joe

67. -----Original Message----From: Dr. Marie [mailto:grame003@umn.edu]
Sent: Friday, June 04
To: Bill
Subject: RE: Submission to arrive tommorrow
Hi Bill ,
I received the case. We will do Flu PCR (4 pools of 4 swabs each), Myco PCR (4 pools
of 4 swabs each), PRRS PCR (4 pools of 4 sera each), and antibody testing for Flu by
a Multiscreen ELISA. I do think testing for PRRS by PCR is a good idea just in case
they came into contact with a new PRRSV post-entry. Skipping the PRRS ELISA for now
b/c we know they've been exposed. I think screening for flu antibodies is a good
idea. If the values I obtain for flu antibodies are strong (s/n values <0.3 for this
competitive ELISA) it would suggest to me that the gilts have been naturally exposed
to flu b/c the vaccine doesn't usually cause as strong of an humoral immune response
detected by ELISA.
Very interesting case. I'll do my best.
Thanks, Marie

71. -----Original Message----From: Bill
Sent: Wednesday, June 09,
To: Marie
Subject: Re: Gilt Respiratory Case
Dear Marie,
Need some more help. On these gilts, they have been
vaccinated with Flu Vax. Are the SIV titers reflective of
this vaccine? If the titers are not reflective of the
vaccine, and since the PCR SIV was negative, is there a way to
use the sera to further delineate the SIV status of the
animals (ie. strain of SIV the gilts have been exposed to)?
Also, the MPS PCR was negative. What is the time window that
the PCR may have been successful in detection? Is this a very
valid test or if the lab still has the sera, would serology be
a good way to test for exposure of these MPS non-vaccinated
animals? It is likely that the exposure was only 3-4 weeks
ago when they entered this positive herd of sows. Any other
suggestions? The group has responded reasonably well to
antibiotics in the water suggesting that MPS is involved since
it is very narrow spectrum.
Looking forward to your comments.
Bill

72. _____________________________________________
From: Dr. Marie
Sent: Thursday, June 10, 2010 11:29 AM
To: Bill
Subject: RE: Gilt Respiratory Case
Hi Bill ,
Sorry for the late reply. I had a vacation day yesterday.
Anyway - here's my interpretations:
•The Flu ELISA results are quite strong and suggestive of animals that have been naturally exposed to flu. Given
that they were also vaccinated, I can't say that with 100% confidence they were infected. What I can tell you is the
vaccinated only pigs (not flu infected) have Flu ELISA S/N ratios around 0.5 to 0.6. The majority of your sera
samples were <0.5 which is what we see with natural infection. Yes, we can tease out specific HI responses by
doing serology against the different vaccine strains in FluVax and some common field strains - that would add to
the cost.
•RE: the Mycoplasma hyopneumoniae PCR tests, there is one pool that we are testing individually - it contains
swabs 33, 34, 35, and 36. As a pool, those swabs were very weakly positive. Perhaps there is an individual
positive that will come out. I believe that testing will be complete either Friday 6-11 or Tuesday 6-15. So, it could
very well be that there is a Mycoplasma hyopneumoniae infection occurring. They Myco PCR on nasal swabs
works well on naïve animals, and can detect shedders in the first few weeks of infection, especially if there are
clinical signs. Definitely wait and see how the individual Myco PCR testing on nasal swabs 33, 34, 35, and 36
comes out to rule in or rule out Myco.
•Yes, I think doing Mycoplasma hyopneumoniae IDEXX ELISA titers is a good idea. Naturally infected animals (if
they were infected greater than 4 weeks prior to sample collection) will have high ELISA s/p values usually >2.0.
In summary, I'm still having a difficult time distinguishing if this group went through a flu infection or myco
infection - it maybe that they had both!
At the very least, let's definitely do the Mycoplasma hyopneumoniae IDEXX ELISA testing. If they are positive on
that, given that they are not vaccinated against Myco, that will be suggestive that they were infected with Myco.
Response to Linco treatment also is suggestive of Myco.
More later, Marie

74. From:
Sent:
To:
Subject:
Bill
Thursday, June 10
Marie
Re: Respiratory Case
Go ahead with the mps serology. Thanks and looking forward
to more comments.
Bill
Sent via BlackBerry by AT&T

77. From: Marie
Sent: Friday, June 11
To: Bill
Subject: RE: Respiratory Case
Hi,
It was Mycoplasma hyopneumoniae
infection. Nasal swab #33 was
positive by PCR. Plus, seropositive in
12 of 16. Good job.
Thanks, Marie

78. -----Original Message----From: Bill
Sent: Monday, June 21
To: Marie
Subject: Respiratory Case.
Marie,
On this case, would you still have any serum left from the pigs to do
PRRS PCR? This farm has blown with PRRS as of last week and it is
suspicious that PRRS may have come along with the gilts, either from
our inoculation or from an outside exposure shortly before the entry.
If you have sera, please do 3-4 pools for PCR PRRS. Let me know.
Thank you,
Bill

79. -----Original Message----From: Marie
Sent: Monday, June 21
To: Bill
Subject: RE: Respiratory Case.
Hi Bill ,
PRRS PCR was completed back on 6-7-10 and all
sera were negative for PRRSV (pools of 4).
Thanks, Marie

80. Can you DX resp dz without
necropsy???
Yes – if you are thorough and
thoughtful and communicate with
the diagnostician

81. Conclusions
•
•
•
•
Pig/Sample selection is important
Sample handling is important
Information sharing is important
Good information and good diagnostics are
worth the investment

82. Teamwork has its sweet rewards!