Increase the Value of Your Diagnostics and Your Value as a Diagnostician

Marie Culhane, DVM, PhD, Jim Collins, DVM, PhD, Dipl. ACVP,

Rebecca Davies, PhDUniversity of Minnesota

Veterinary Diagnostic Laboratory St. Paul, MN

$ 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?

Presentation Overviewand Steps to Improve your Diagnostic Value

• Proper sampling technique • Submission of samples• Interpretation of results

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

How do you get there?

Do a proper disease investigation!

Starting a Disease Investigation

• What Problem are you Investigating?– Clinical signs?– Unexpected/undesired serology results or

slaughter check findings– Production changes

PreparationPig selection

TimeThermometerClinical ObservationEuthanized vs. deadTreated vs. Untreated

Disease Investigation

Disease Investigation

• Necropsy– Tools– Assistants– Euthanasia method– Environment– Begin

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

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

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

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

Cecal contents

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

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 calcium-alginate-tipped swabs have been reported.

• One inexpensive, multi-purpose swab is BBL CultureSwab Liquid Stuart, Single Swab (pkg of 50), Product Number 220099.

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”

Specimen Submission

• Outer bag, labeled– Protect shipping container

• Submission form– Outside of cooler, in cardboard box

• Same day or overnight delivery

Sample handling still counts and still costs!

Rebecca L Davies, PhDQuality Assurance Coordinator

Veterinary Diagnostic LaboratoryUniversity of Minnesota

We want to get it right the first time.

High degree of accuracy = minimal number of errors

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

Errors in Laboratory Medicine

Categories of error

– Pre-analytical phase (Farm / Clinic)– Analytical phase (Laboratory)– Post-analytical phase (Shared responsibility)

Pre-analytical phase errors

• Animal status and information sharing• Specimen collection• Specimen processing• Specimen transport• Specimen delivery to instrument for analysis

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

Post-analytical phase errors

• Reviewing results for discordance• Interpretation and utilization of results• Appropriateness of reference intervals• Post analysis sample storage

Errors in Laboratory Medicine

Categories of error

–Pre-analytical (Clinic + Laboratory)–Analytical (Laboratory)–Post-Analytical (Clinic + Laboratory)

Pre-analytical procedures: Sample handling hoops

Sample labelingTube typeTube fill levelStorage time/tempTransport time/tempCondition received at lab

Animal Status and Information Sharing Errors: Clinical Examples

• Reason for Submission– Diarrhea

• Age of Pig– 3

• Sample– Feces

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.

Specimen Collection Errors: Clinical Examples

• Reason for Submission– Pale pigs

• Age of Pig– 2 wks

• Sample– ???

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

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.

Minnesota Veterinary Diagnostic Laboratory

Stability of PRRSV in Laboratory Settings

Stability of PRRS in Serum

0

2

4

6

8

10

12

14

16

0 20 40 60 80 100 120 140 160 180

Time (hours)

vR

NA

Qty

.

25 degrees

4 degrees

Cut off = 0.789cutoff

Stability of Flu RNA in media

0

5

10

15

20

0 24 48 72 96 120Time (hrs)

Qty

of

vRN

A

37 C 25 C 4 C

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

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.

Which errors occur with the greatest frequency?

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”.

A look at the literatureErrors 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 pre-and post-analytical phases, with fewer mistakes (13-32%) occurring during the analytical phase”

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 resultsFound 189 errors (0.47%)

68% were pre-analytical13% were analytical18% were post-analytical

Reducing Laboratory Errors

A Shared Responsibility

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

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

Example of Proper Disease Investigation

• Prioritize your differentials and/or work with the laboratory to decide which tests to do first

Prioritize your Differentials

• Which diseases are most important to rule out or rule in

• Which tests can be done the most rapidly

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 typedays

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

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

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 = 119841HTwo sets of fixed and unfixed pig tissues with IDs as follows: – 6 = U4459(coughing) – 7 = Q448 (no clinical signs)

• 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.

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).

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.

An Example of an Incomplete (albeit still useful and diagnostic)

Disease Investigation Example

A Case Study

Can you DX resp dz without necropsy???

-----Original Message-----From: BillSent: Thursday, June 034:35 PMTo: MarieSubject: Submission to arrive tommorrowDr. 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

-----Original Message-----From: Dr. Marie [mailto:grame003@umn.edu] Sent: Friday, June 04To: Bill Subject: RE: Submission to arrive tommorrowHi 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

-----Original Message-----From: BillSent: Wednesday, June 09, To: Marie Subject: Re: Gilt Respiratory CaseDear 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

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From: Dr. Marie Sent: Thursday, June 10, 2010 11:29 AMTo: Bill Subject: RE: Gilt Respiratory CaseHi 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

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

From: Marie Sent: Friday, June 11To: 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

-----Original Message-----From: Bill Sent: Monday, June 21To: MarieSubject: 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

-----Original Message-----From: Marie Sent: Monday, June 21To: 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

Can you DX resp dz without necropsy???

Yes – if you are thorough and thoughtful and communicate with

the diagnostician

Conclusions

• Pig/Sample selection is important• Sample handling is important• Information sharing is important• Good information and good diagnostics are

worth the investment

Teamwork has its sweet rewards!