'Biochemical analysis of non- standard fluids: clinical ...

'Biochemical analysis of non-standard fluids: clinical and analytical considerations.

Presentation by:

Dr Peadar McGing, FRCPath EuSpLM, Principal Clinical Biochemist, Department of Clinical Chemistry and Diagnostic Endocrinology, Mater Misericordiae University Hospital, Eccles Street, Dublin 7, D07R2WY, Ireland.

Biochemical analysis of non-standard fluids - Focus 2018 - Dr Peadar McGing

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My first fluid testing case

• 12 year old male patient

• Acute pains lasting only seconds in different part of abdomen.

• Increased frequency of pain.

• Multiple negative blood and radiology tests (over 3 weeks as in-patient).

• Lumbar puncture performed – CSF tests negative.

• What next?


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My first fluid testing case

• What next? Pain frequency decreased post LP

• Second LP performed. Pain disappeared

• ?Long term outcome.

• Patient is currently 50 years symptom free.


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5 Biochemical analysis of non-standard fluids

- Focus 2018 - Dr Peadar McGing


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“Instruction does much, but encouragement everything.”

– Johann Wolfgang von Goethe, writer and statesman

Lecture Outline

• Reference Books

• Body fluids

– Types of fluids

– Some physiology and pathology

• Pre-analytical

• Analytical

• Post-analytical


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• Copyright: 2015; ISBN: 9781594251801

• Pages: 56; Price: $22.00; Member Price: $18.00



From ACB Mailbase


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Clinical Significance

Sodium = 125 mmol/L

versus

Sodium = 141 mmol/L

• Is there a clinical difference in these results?


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Clinical Significance

Sodium = 125 mmol/L

versus

Sodium = 141 mmol/L

• Is there a clinical difference in these results?

• YES if serum / plasma

• NO if urine


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Let’s talk about fluids

• Fluid formation

• Fluid function

• Fluid accumulation

• Fluid removal

• Fluid analysis

Biochemical analysis of non-standard fluids

- Focus 2018 - Dr Peadar McGing 16

Types of Fluids for Analysis

• Cerebral Spinal Fluid

– Query CSF

• Pleural Fluid

• Pericardial Fluid

• Ascitic / Peritoneal Fluid

– PET Dialysis Fluid

• Saliva

• Sweat

• Amniotic Fluid

• Seminal Fluid

• Synovial Fluid

• Cyst Fluids

– Pancreatic cyst fluid

• Faecal Water

• Vitreous humour

• Drain fluids


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The age-old question


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fluids

Body Fluids

• Body fluid / body water – c60% of body wt

• Two-thirds Intracellular Fluid (ICF)

• One-third Extracellular Fluid (ECF)

• One quarter of ECF is Intravascular (plasma)

• Three quarters is Extravascular.


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Origin of Fluids

• Body fluid / body water – c60% of body wt Two-thirds Intracellular Fluid (ICF), One-third Extracellular Fluid (ECF)

• One quarter of ECF is Intravascular (plasma), three quarters Extravascular.

• Extravascular fluid:

– Three quarters interstitial fluid

– One quarter transcellular fluid

• Separated from plasma by additional epithelial layer

• Incl CSF, pleural fl, pericardial fl, peritoneal fl, synovial fl, intraoccular fl, …


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Fluid collection for testing

• Blood readily available, but minimally invasive,

• Urine can be readily available (timing-dependent)

• Transcellular fluid may be difficult – – Increasingly Ultrasound guided,

– Sometimes just diagnostic tap

– Often fluid drainage is therapeutic


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Body Fluids

• Fluid function: – Protection (e.g. CSF, amniotic fluid)

– Waste removal (e.g. CSF)

– Lubrication (e.g. pleural, pericardial fluid)

• Fluid accumulation: – Increased production

• Raised hydrostatic pressure (e.g. heart failure),

• Increased permeability (e.g. infection, malignancy)

– Decreased removal


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Pleural Effusion

• “Pleural fluid is not just there to annoy respiratory physicians but has a purpose. It allows us to breathe.”

– Opening remark of Consultant Respiratory Physician at Medical Grand Rounds presentation ‘A fluid situation. Myths, pearls, and reality of pleural effusions’.


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Pleural Effusion

• Pleural effusion → fluid formation exceeds removal → accumulation of excess fluid in the pleural space.

• Fluid accumulation can be due to – increased fluid production or – decreased fluid removal.

• Etiology is related to the underlying condition • – congestive cardiac failure causes increased fluid due

to raised hydrostatic pressure gradient • – malignancy or infection, the increased production is

usually the result of increased permeability of capillary vessels.


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Pleural fluid- physical characteristics

Fluid Appearance/ Odour

Necessary Fluid Study

Differential

Bloody Hematocrit and RBC count

Malignancy, trauma, PE, hemothorax

Cloudy Triglycerides Chylothorax

Putrid odour Gram stain and culture

Anaerobic infection


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Fluid appearance



Pleural Effusions

• The most common causes of exudative pleural effusions are – parapneumonic effusions (particularly bacterial

pneumonia), and

– malignancy.

• The most common causes of transudative pleural effusions are – left ventricular failure (very common) and

– cirrhosis.


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Lapworth R and Tarn AC. Commentary on the British Thoracic Society guidelines for the investigation of unilateral pleural effusion in adults. Ann Clin Biochem 2006; 43: 17-22.

Transudate v Exudate

• Fluid Protein strongly suggests

Transudate if Fluid Protein <25 g/L

Exudate if Fluid Protein >35 g/L

• Fluid Protein equivocal if 25-35 g/L

Further testing required, including Fluid LDH, or

Light’s Criteria


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Clin Chest Med 34 (2013) 21-26.

Light’s criteria (Pleural Fluid)

• Pleural fluid is an exudate if one or more of the following criteria are met:

– Pleural fluid protein divided by serum protein is >0.5

– Pleural fluid lactate dehydrogenase (LDH) divided by serum LDH is >0.6

– Pleural fluid LDH >2/3 the upper limits of laboratory normal value for serum LDH.


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Pleural Effusions

• Where clinical suggests transudate but Light Criteria suggest exudate:

• Serum:fluid protein gradient >31 g/L suggests Transudate,

• If protein gradient <31 g/L then

– Fluid NT-Pro BNP >1300ng/L, or

– Serum:fluid Albumin gradient >12 g/L

suggests Transudate.


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CONCLUSIONS: Regardless of the daily drainage, chest tubes can safely be removed earlier than anticipated in most patients after lobectomy if the protein content of the draining fluid is low. [Def’n of Low: Drains were removed if the pleural fluid to blood protein ratio (PrRPl/B) was ≤0.5.]


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Pleural Fluid Report


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Reference ranges are not reported for fluid biochemistry. Fluid biochemistry should be interpreted in light of the clinical question being asked, comparison with blood results, and/or appropriate clinical guidelines.

Biochemical Tests performed in specific clinical circumstances.

• Query Chylothorax

– triglycerides and cholesterol +/- chylomicrons

• Query TB

– (Adenosine deaminase)

• Query Malignancy

– value of tumour markers is questionable.

• Query Pancreatitis

– amylase.



Patient A.

• b/g end-stage ischaemic cardiomyopathy.

• Chest drain inserted

• Sample: Pleural Fluid

• Test requested: TRIGFL (Fluid Triglyceride)

• Clinical Details: Query chylothorax

• Result: TRIGFL = 4.01 mmol/L

• Query re interpretation.

• Biochemist added CHOLFL (Fluid Cholesterol)

• Result: CHOLFL = 1.40 mmol/L Biochemical analysis of non-standard fluids


Interpretation

Chylothorax Pseudo-chylothorax

Trig (mmol/L) >1.24 <0.56

Chol (mmol/L) <5.18 >5.18

Fl to Ser Chol Ratio <1

Fl to Ser Trig Ratio >1

Chylomicrons Present Absent

Cholesterol Crystals Not seen Often seen


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Ref: adapted from McGrath et al. Respiratory Medicine (2010) 104, 1-8.

Patient A - Interpretation

Chylothorax Pseudo-chylothorax

Trig (mmol/L) >1.24 <0.56

Chol (mmol/L) <5.18 >5.18

Fl to Ser Chol Ratio <1

Fl to Ser Trig Ratio >1

Chylomicrons Present Absent

Cholesterol Crystals Not seen Often seen


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TRIGFL = 4.01 mmol/L CHOLFL = 1.40 mmol/L

Clinical Audit (Bio / Respiratory)


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Audit findings

• 34 consecutive pleural fluids


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Fluid test n %

Culture 32 94

LDH 29 85

Gram stain 29 85

Protein + albumin 27 79

pH 26 76

Glucose 21 62

Cytology 14 41

Serum / Plasma n %

Blood with fluid 17 50

Protein + albumin 14 41

LDH 8 24

Glucose 7 21

Pre-analytical

• The correct sample

• Correctly labelled – Including fluid source

• All tests requested – Including paired blood tests

• All test samples taken – and in correct tubes

• All samples presented in timely manner

• All samples processed in timely manner


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Pre-analytical

• Some examples of MMUH communications and procedures aimed at reducing pre-analytical issues


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Fluid Analysis

The Following Sample Containers are required for Collection of Fluid

(Pleural/Ascites/Peritoneal) to ensure appropriate Test Analysis

Product No: 75.9922.745. 70ml MSU container Laboratory: Microbiology Test Request: FL C/S & Gram stain; FL ZN/TB Culture (Pleural Fluid only)

N. B. Please send separate Pleural Fluid sample for each test request -------------------------------------------------------------------------------------------------------

Product No: 05-1167-001. 2.7 ml EDTA KE Pink capped tube OR

Product No: 05.1167.901. 2.7 ml EDTA KE Grey capped tube Laboratory: Microbiology Test Request: Fluid Cell Count Mix well by inverting gently 4-5 times -------------------------------------------------------------------------------------------------------

Product No.75.9922.745 70ml MSU Laboratory: Cellular Pathology Test Request: Diagnostic Cytology N. B. Please do not add any fixative to this sample. Deliver fresh to lab ASAP. 50mls aliquot deliver as soon as possible. Place 3 IU heparin per ml of fluid to prevent clot formation

46 Biochemical analysis of non-standard fluids - Focus 2018 - Dr Peadar McGing

Mater Misericordiae University Hospital MI-GEN-011 Pathology Laboratory Edition 1.03

Product No: 04.1940.001. 4.9 ml ORANGE capped Lithium-Heparin gel Tube Laboratory: Clinical Chemistry & Diagnostic Endocrinology Test Requests [Routine]: Fluid Protein(PROTFL), Fluid Albumin(ALBFL), Test Requests [Occasional]: Fluid Cholesterol(CHOLFL), Fluid Triglycerides(TRIGFL), Fluid Amylase(AMYLFL), Fluid Bilirubin(BILIFL), Fluid CA125(CA125F), Fluid Urea(UREAFL), Fluid Creatinine(CREAFL), Fluid CA19.9(CA199F), Fluid CEA(CEAFL) Mix well by inverting gently 4-5 times N. B. Please ensure blood sample is also collected -------------------------------------------------------------------------------------------------------

Product No: 04.1935.001. 4.9 ml BROWN capped Serum gel Tube Laboratory: Clinical Chemistry & Diagnostic Endocrinology Test Request: Fluid LDH (LDHFL) Mix well by inverting gently 4-5 times N. B. Please ensure blood sample is also collected -------------------------------------------------------------------------------------------------------

Product No: 04-1903-001. 2.6 ml Fluoride EDTA YELLOW capped tube. Laboratory: Clinical Chemistry & Diagnostic Endocrinology

Test Request: Fluid Glucose (GLUFL) Mix well by inverting gently 4-5 times N. B. Please ensure blood sample is also collected ---------------------------------------------------------------------------------------

Product No: 956-622. safePICO Aspirator Arterial Blood Sampler Laboratory: Clinical Chemistry & Diagnostic Endocrinology: Test Request [Routine]: Fluid pH (PHFL), Test Request [Occasional]: Fluid Lactate (LACTFL) Mix well by inverting gently 4-5 times

47 Biochemical analysis of non-standard fluids - Focus 2018 - Dr Peadar McGing

Pleural fluid sampling instruction. -for clinical staff, available on hospital intranet


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Laboratory guide to fluid sample types Mater Misericordiae University Hospital LI-CCE-0051 Clinical Chemistry and Endocrinology Laboratory Edition 1.02

Clinical Chemistry/Endocrinology - Processing Chart for Fluids This refers to Fluid samples only; refer to LI-CCE-0012 and LI-CCE-0050 for Blood and urine processing charts respectively

Subscript numbers refer to Tube type e.g. 2 is Brown serum gel tube.

TEST Telepath Code Tube Type Fluid type allowed for assay (for entering as specimen type on Telepath)

CSF Protein CPROT Plain aliquot CSF

CSF Glucose CGLU Plain aliquot CSF

CSF Xanthochromia XAN Plain aliquot protected from light

SF2

Fluid Protein PROTFL Orange Top Gel PF, PER, PAF, PC, FL

Fluid Albumin ALBFL Orange Top Gel PF, PER, PAF, PC, FL

Fluid LDH LDHFL Brown Top Gel PF2, PER2, PAF2, FL2

Fluid Cholesterol CHOFL Orange Top Gel PF, PER, PAF, PC, FL

Fluid Triglycerides TRIGFL Orange Top Gel PF, PER, PAF, PC, FL

Fluid Amylase AMYFL Orange Top Gel DR, PAF, FL

Fluid Bilirubin BILIFL Orange Top Gel DR, FL

Fluid CA125 CA125F Orange Top Gel PF, PER, FL

Fluid Urea UREAFL Orange Top Gel PET, FL

Fluid Creatinine CREAFL Orange Top Gel PET, DR, FL

Fluid CA199 CA199F Orange Top Gel PAF, FL

Fluid pH PHFL Blood Gas Monovette PF4, PER4, PAF4, FL4

Fluid Lactate LACTFL Blood Gas Monovette DR4, CSF, FL4

Fluid Glucose GLUFL Yellow Top PF3, PER3, PAF3, FL3, PET3, DR3

Fluid CEA CEAFL Orange Top Gel PF, PER, PAF, FL

Stability: CSF XAN – see special protocol. All other tests: stability as for plasma / serum, once in correct containers.

Fluid Types Code

CSF (Cerebral Spinal Fluid) CSF

CSF for Xanthochromia SF2

Pleural Fluid PF

Ascitic / Peritoneal Fluid PER

Dialysis Fluids PET

Fluid Types Code

Drain Fluids DR

Pancreatic Cyst Fluids. PAF

Pericardial Fluids PC

All other Fluids FL



Fluids SOP

Procedure for Body Fluids other than Blood and Urine

Mater Misericordiae University Hospital

REVISION DESCRIPTION

Added comment re use ‘off-label’ (1.0) Added re salivary cortisol (1.0, 2.0, 3.4) Added section on Stability (3.3) [CR5018] Added Pericardial Fluid as specimen type (4.0, 5.0) Re-formatted pages.



Fluids – Pre-analytical

• 3.3 Stability

• Provided samples are collected into appropriate tubes, or transferred to such tubes soon after collection (e.g. Fluoride EDTA for fluid glucose), then stability is the same as for that test in plasma. For stability times see LI-CCE-0009 [ANALYTE STABILITY CHART].

• CSF for glucose should be analysed within one hour if significant cells present.


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Procedure for Body Fluids other than Blood and Urine

Mater Misericordiae University Hospital

REVISION DESCRIPTION

Added comment re use ‘off-label’ (1.0) Added re salivary cortisol (1.0, 2.0, 3.4) Added section on Stability (3.3) [CR5018] Added Pericardial Fluid as specimen type (4.0, 5.0) Re-formatted pages.

Most testing described in this SOP is ‘off-label’, i.e. testing is carried out in a fluid not validated by the manufacturer. All reports carry a statement to this effect.



Email to ACB mailbase, Jan 2016 Subject: UKAS accreditation for pleural and ascitic fluid

• ‘It is my understanding that measurements of certain analytes in fluids such as ascitic and pleural fluid cannot be UKAS accredited for several reasons including CE marking, lack of fluid EQA, standardisation issues in non-recommended matrix etc. These include: albumin, amylase, bicarbonate, calcium, creatinine, glucose, LDH, potassium, sodium, triglyceride, total protein and urea in pleural and ascitic fluid …. ’

• ‘We let our users know that these are non-accreditable tests.’

• ‘Is anyone tempted to drop them due to UKAS, or not bother with UKAS for fluids or has anyone overcome this by local verification?’


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Email to ACB mailbase, Jan 2016 Subject: UKAS accreditation for pleural and ascitic fluid

• Feedback:

• ‘The replies can be split into 2 groups:’

• ‘We have removed these from our AC6. Seems the easiest way to deal with the issue’

• ‘Just go for "this report is not UKAS-accredited“.’

• Concern:

• ‘We are now in the realm of having “UKAS accredited tests” and “non-UKAS Accredited tests”, which is an accreditation place that I did not want to be. For example once we accept that we are going to provide a clinical service with some “non-UKAS accredited tests” where do we draw the line?’


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The inspector?


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The fluids biochemist?


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– Query CSF

• Pleural Fluid




• Saliva • Sweat

• Amniotic Fluid

• Seminal Fluid

• Synovial Fluid

• Cyst Fluids


• Faecal Water

• Vitreous humour

• Drain fluids


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Ascitic / Peritoneal Fluid

• Common clinical finding – wide range of causes.

• Conditions that may be associated with ascites include – increased hydrostatic pressure associated with portal

hypertension (e.g cirrhosis, alcoholic hepatitis) – decreased colloid osmotic pressure secondary to

hypoalbuminaemia (e.g. ESLD, nephrotic syndrome) – increased permeability of peritoneal capillaries (TB

peritonitis, HIV peritinotis) – leakage of fluid into the peritoneal cavity (e.g. bile, urine) – malignant conditions (e.g. peritoneal carcinomatosis) – miscellaneous causes (e.g. myxoedema, chronic HD)



SAAG

• SAAG = [S-Alb]– [F-Alb]; all in g/L

• SAAG greater or equal to 11 predicts that the patient has portal hypertension with 97% accuracy.

• Runyon BA1, Montano AA, Akriviadis EA, Antillon MR, Irving MA, McHutchison JG. The serum-ascites albumin gradient is superior to the exudate-transudate concept in the differential diagnosis of ascites. Ann Intern Med. 1992 Aug 1;117(3):215-20.


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Remember effect on serum assays

• Type of comment I sometimes add to report of raised plasma CA125 in patient with fluid accumulation –

Noted receipt of peritoneal fluid for analysis, indicating presence of ascites. Ascites can of itself cause marked rise in plasma CA125 (to higher than level seen in this case).

Interpret with caution.

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• “Your tests reveal you are retaining fluids.”



Could this fluid be leaked CSF?

• Pt B.

• Male in late 50s.

• Fluid noted on scan, and removed

• Surgeons keen to know origin of fluid before doing exploratory operation.

• Could be CSF

• What test?


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• Pt B.

• Male in late 50s.

• Surgeons keen to know origin of fluid before doing exploratory operation.

• Could be CSF

• What test?

• Measure Beta Trace Protein – We use Health Services Laboratories in London

• Beta Trace Protein now preferred to Beta-2-Transferrin (Asialotransferrin)


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• Measure Beta Trace Protein (BTP) • Fluid:

– appearance = straw coloured – BTP = 1.61 mg/L (ref: <1.30 = negative, 1.31-8.88

=intermediate positive, >8.89 = raised positive) – Suggests possible CSF leak

• Serum: – BTP = 0. 61 mg/L (ref: <1.30 = normal)

• Fluid to serum ratio: – Ratio = 2.63 (>2.0 indicates probably fluid is CSF).


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Medical Grand Rounds - case presented by Consultant Endocrinologist

• Patient C: 59 yrs male

• May 2012 to Apr 2016 had 6 admissions

• Adm Apr’16 w drowsiness + ataxia.

• Hx included Hashimoto’s thyroiditis and left hemi-thyroidectomy.

• Adm bloods – TFTs=N (FT4=18.2, TSH=2.19), Anti-TPO=38.5 (ref<5); mild anaemia.

• ?EATTD (Encephalopath Autoimmune Associated Thyroid Disease)


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• 59 yrs male with possible diagnosis of EATTD (Encephalopath Autoimmune Associated Thyroid Disease).

• Exclude other causes of encephalopathy

• Test that could maybe be helpful

CSF Anti-TPO

• But is the assay validated / verified for CSF?


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• As our assay was not validated Neurology Dept had sent CSF to USA for TPO analysis

• Result not available

• Deemed clinically that dx did = EATTD

• Post Grand Round, I decided to do very basic verification


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TPO mini-verification

• Samples used:

• CSF – old samples from freezer storage (next samples due for routine disposal); anonymised.

• Plasma – aliquots of samples from routine TPO assay; TPO<1 or TPO>200 IU/mL.

• Test sample: 50µL plasma + 950 µL CSF


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TPO mini-verification

• Plasma – aliquots of samples from routine TPO assay; TPO<1 or TPO>200 IU/mL.

• Test sample: 50µL plasma + 950 µL CSF


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CSF TPO CSF+(TPO<1) (TPO<1) Expected Recovery

1 0.83 0.86 0.24 0.80 107.4%

2 0.00 0.00 0.24 0.01 NA

3 0.00 0.00 0.24 0.01 NA

TPO CSF TPO CSF+(TPO pos) (TPO pos) Expected Recovery

1 0.83 43.11 756 38.59 111.7% 2 0.00 44.94 756 37.80 118.9% 3 0.00 37.88 756 37.80 100.2% 4 0.83 70.33 1246 63.09 111.5% 5 0.00 63.83 1246 62.30 102.5% 6 0.00 63.64 1246 62.30 102.2% 7 0.83 12.14 209 11.24 108.0% 8 0.00 10.55 209 10.45 101.0% 9 0.00 10.74 209 10.45 102.8%

mean 106.5% SD 6.4%


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Tamagno et al. BMC Neurology 2010, 10:27

In the CSF, anti-TPO and anti-TG Abs were positive in 5/5 (Patient 2,

6, 7, 10, and 14) and 2/2 (Patient 6 and 7) cases, respectively.

The challenge of fluid analyses


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“In life, there is always a hurdle.”

– Gail Devers, track and field athlete

• Photo courtesy of IAAF

Safety

• Options – Reject – Treat as do for plasma / serum/urine – Prepare in fume cupboard – Use special category high-risk fume cupboard

• Sample dependent • Local availability of facilities • Safety considerations for in-house AND for

referral • Rem CSF for Xanthochromia


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MMUH Fluids SOP

• 6.2 External QA • Other than for CSF (and the schemes for blood and

urine) there is no EQA scheme covering analysis of biochemical parameters in Body Fluids.

• Details of the EQA scheme for CSF are to be found in the SOP for EQA [QP-CCE-0001]

• CSF Protein is also specifically covered by RIQAS Urine Scheme (as well as CSF-specific scheme).

• We use the EQA schemes for blood and urine to monitor quality of performance for all other tests carried out on miscellaneous body fluids.


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EQA of Fluids - Options

• EQA of specific fluid

– Plasma / serum

– CSF

– Other

• Assurance from plasma/serum

– Testing the analyte

– Testing the system

• EQA by sample exchange


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Glucose EQA

• MMUH Routine Glucose assay = Hexokinase/G-6-PDH (on Abbott Architect)

– EQA in Plasma / Serum – Good

– EQA in Urine – Good

– EQA in CSF – Good

• MMUH POCT Glucose assay = Glucose sensor (electrode) (on ABG machine)

– EQA in Whole Blood – Good


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Lactate EQA

• MMUH lactate assay = Lactate sensor (electrode) (on ABL90 ‘blood gas’ machine)

– EQA in Whole Blood – Good

– EQA in Plasma / Serum – Good

• Used CSF EQA as initial test of validity of our Lactate assay if applied to CSF.

– EQA in CSF – Poor Decision = refer CSF Lact



Saliva

• Mouth fluid possessing several functions

– Oral health and homeostasis

– Taste perception

– Oral mucosa protection against mechanical damage

– α-amylase

– Teeth enamel mineralisation

– Defence functions

• Mouth fluid as test medium

– Significant Advantage….non-invasive collection

– Free hormones

Slides on Salivary Cortisol method courtesy of Keith Mulready, MMUH.

Production and composition

• 500 – 1500 ml/day

• Modified by several physiological and pathological influences

• Autonomic nervous system controls output and composition

• Serous…sympathetic nervous system

• Mucous…both para- and sympathetic

• Clearance of compounds from plasma into saliva involves

– Ultrafiltration…only molecules MW<1900 Da involved • Small polar molecules

– Transudation • Albumin

– Selective transport through cellular membranes • Passive diffusion of lipophilic molecules

• Free, non-protein bound unconjugated steroids

Slides on Salivary Cortisol method courtesy of Keith Mulready, MMUH

Cortisol analysis by Immunoassay

CBG

80-90%

Albumin

10-15%

Free Cortisol

5-10%

• Large intra + inter-platform variability

• New Roche II 25% lower than Roche I

• Numerous CRMs in use • Diff antibodies to different

epitopes • No consensus for diagnostic

cut-offs • Displacement steps are

manufacturer specific Use of another steroid, a change

in pH or use of a high affinity antibody to compete for binding

Cortisol Transport + Binding (Plasma)

Slides on Salivary Cortisol method courtesy of Keith Mulready, MMUH

LC-MS/MS is 10-100 times more sensitive when compared with IA-based methodology and is capable of eliminating many of the inherent

flaws encountered with immunoassays

Metrological Traceability

Need to fulfil the requirements of the IVD directive and ISO15189

Minimum of 6 certified reference materials for Cortisol ERM-DA 192 and 193

ERM-DA 451/IFCC

SRM 921

SRM 971

U.S. Pharmacopeia Hydrocortisone Reference Standard

NIST standard reference material 921 was used for cortisol calibrators

Cerilliant CRM for calibrators and IQC Cortisone

The traceability chain is maintained

Validation

• FDA Guidance for Industry Bioanalytical Method Validation

• Honour, J.W., 2011. Development and validation of a quantitative assay based on tandem mass spectrometry. Annals of Clinical Biochemistry, 48(Pt 2), pp.97–111.

Assessment

Intra-precision

Inter-precision

Accuracy

Post Extract Stability

Injection Precision

Ion Suppression

(Post Column Infusion)

Carryover

LC-MS/MS Referral

Peer Comparison

Linearity

Analyte Recovery

Dilutional Studies

Sample stability Slides on Salivary Cortisol method courtesy of

Keith Mulready, MMUH


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– Query CSF

• Pleural Fluid




• Saliva

• Sweat

• Amniotic Fluid

• Seminal Fluid

• Synovial Fluid

• Cyst Fluids


• Faecal Water • Vitreous humour

• Drain fluids


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‘Father of Microbiology’


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Antonie van Leeuwenhoek (1632 – 1723)

• Dutch merchant

• First to see microorganisms (‘animalicules’),

• First to see Sperm, and describe fertilisation,

• First to describe Urate crystals.

• [Figure from original drawing]


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Synovial Fluid

• Gold standard for diagnosis of gout is identification of MSU (monosodium urate) crystals in synovial fluid.

• Differentiate MSU from CPPD (calcium pyrophosphate dihydrate) crystals.

• Polarised light microscope is required. – MSU crystals have negative birefringence (rotate

beam of polarised light clockwise).

– CPPD crystals have positive birefringence (rotate beam of polarised light anti-clockwise).



Pancreatic Cyst Fluid

• Pancreatic cyst fluid analysis for differential diagnosis between benign and malignant lesions.

• Renata Talar-Wojnarowska et al. Oncol Lett. 2013 Feb; 5(2): 613–616


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Pancreatic Cyst Fluid


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Copyrights apply

Copyrights apply

Typical carcinoembryonic antigen (CEA) level: <5 to 20 ng/mL in majority of lesions. Relative malignant potential: Negligible

Typical carcinoembryonic antigen (CEA) level: >200 ng/mL in approximately 75% of lesions. Relative malignant potential :Moderate, High, Low to moderate respectively

Faeces / Faecal Water

• Faecal calprotectin

• Faecal elastase

• Faecal electrolytes

– Sodium

– Potassium

– Magnesium

• Faecal Osmolality


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Copyrights apply

Remember testing may not be always appropriate.

Biochemical analysis of non-standard fluids - Focus 2018 - Dr Peadar McGing 103

Summary (1)

• Many body fluids can accumulate in disease

• In some cases laboratory testing, including biochemical, may be of value.

• Testing of other fluids (not pathological accumulations) can also be helpful.

• The number of samples and analytes so tested is a very small part of our workload

• Despite this, fluid analyses can be difficult and labour intensive.


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Summary (2)

• Matrix differences versus serum/plasma/urine means some verification needed

• Full verification / validation is probably not clinically necessary (usually),

• Matrix effect MUST be tested.

• Some interpretation should be provided either on report, via electronic link, or via reference to hospital intranet /internet.


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Summary (3)

• Message that test is used off-label must be attached to report (unless not appropriate)

• The difficulties associated with verification of fluid analyses should not deter laboratories from using them.

• Biochemists should – carry out verification of any assays likely to be of

value, where possible, – Refer tests only if referral lab has verified assay, – Refuse to assay where could mislead – Refuse to assay where cannot stand over result.


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