Basic Principles of Laboratory Medicine

Dr R. SirkarChemical Pathology Department

the curricula of most medical schools do not emphasize laboratory medicine sufficiently

graduates often enter future practice with major gaps in this area of diagnosis and therapeutics.

laboratory medicine is diverse, most of the critical concepts apply to all of the various subspecialties.

Understanding the appropriate uses, benefits, pitfalls, and shortcomings of clinical laboratory test results ensures their proper use and interpretation;

lack of this knowledge is fraught with risks.

Basic Principles

Not all tests are ordered for the same reason.

Diagnosis

Hospitalization

Surgery

Monitor treatment

Preventative – risk

Use of laboratory test for different purposes

The most sensitive test should be used first.

Stepwise

Cost factors

HIV and Hepatitis screening

Excessive laboratory tests can cause iatrogenic anemia

Neonates

Standing orders

Risks vs benefit vs true need for a particular test

Many laboratory test names are misleading or confusing.

HandwritingEg GGT and GTTHep B surface AG and Anti hep B surface Ag

Tests should be ordered as routine except in an absolute emergency

Abuse of STAT orders may cause more harm than good

Lab staff eventually treat those orders as routine – risk a STAT request is truly required

The time of collection is important:

Cortisol

FSH/ LH

Triglycerides

GTT

The correct collection tube must be used for specimens.

Sample rejection prevents erroneous results.

Hemolyzedspecimen

Clotted specimen

Mislabeled or unlabeled specimen

Quantity not sufficient

Laboratory tests can measure something directly or indirectly

DIRECT eg. electrolytes

Indirect eg. LDL cholesterol

Laboratory tests must be interpreted in light of the clinical situation.

Reference ranges are not universal.Encompasses 95% - 2,5% of normal above and 2,5% normal below rangeDeveloped in adult populations often

Report units may vary. SI units

High and unexpected abnormal results may be spurious

Therapeutic interventions affect test results.

Special test results may return after patients are discharged.

Laboratories have a mechanism to notify clinicians of

critical results- life-threatening values (LTV)

Examples of critical values

Test Low High

pH <7.25 >7.55

Sodium < 120 > 160 Potassium <3.0 >6.5 Lactic acid None >8.0 mmol/L

Urine protein None 4+

HIV Western blot N/A PositiveBlood culture N/A Positive

Pathologists are available for consultation.

Thank you for your time

Vitamin D

Dr R SirkarChemical Pathology

Greys Hospital

Introduction

Rickets – tip of the Vitamin D deficiency iceberg

Vitamin D deficiency in adults exacerbates osteopaenia and osteoporosis, causes osteomalacia, muscle weakness and increased risk of fractures

Vitamin decreases the risk of many chronic illnesses including common cancers, autoimmune disease, infectious diseases and CVD

Sources of Vitamin D

Sunlight

Diet: Natural or fortified foods

Dietary supplements

Metabolism of Vitamin D and its relationship to Calcium

Holick M. N Engl J Med 2007;357:266-281

Definition and prevalence of Vitamin D Defiency

No consensus

Most experts 25 hydroxyvitamin D < 50 nmol/L

Vitamin D intoxication > 374nmol/l

1 billion people worldwide have Vitamin D deficiencey or insufficiency

Osteoporosis and fractures

Evaluation of exclusive use of calcium or vitamin D3 (RECORD trial) showed no antifracture efficacy for patients reciving800IU of vitamin per day.

Muscle strength and falls

Vitamin D deficiency causes muscle weakness

Skeletal muscles have a vitamin D receptor

and may require Vitamin D for maximum function

Non Skeletal actions of Vitamin D

Brain, prostate, breast and colon tissue as well as immune cells have a vitamin D receptor and respond to 1,25 dihydroxyvitamin D

1,25Vit D is also a potent immunomodulator--- Tuberculosis

Vitamin D and Chronic diseases

Cancer

Vit D def associated with a 30 – 50% increased risk of incident colon, prostate and breast cancer along with higher mortality from these cancers

Autoimmune diseases, osteoarthritis and diabetes

Increased risk of type 1 diabetes, multiple sclerosis and Crohn’s disease

Cardiovascular disease

Increased risk of hypertension and cardiovascular disease – congestive heart failure

Schizophrenia and depression

Linked to increased incidence of these disorders

Causes of Vitamin D deficiency

Reduced skin synthesis

Decreased bioavailability

Increased catabolism

Breast feeding

Decreased synthesis of 25 hydroxy vitamin D

Increased urinary loss of 25 hydroxy vitamin D

Decreased synthesis of 1,25 Vitamin D

Heritable disorders

Acquired disorders

Vitamin D requirements

Institute of Medicine recommends:Children 200 IUAdults up to 50yrs 200 IUAdults 51 – 70 years 400 IU

Most experts agree if not enough sun exposure – 800 to 1000 IU per day recommended

Vitamin D requirements

Lactating women need 4000 IU per day

Conditions in which supplementation may need to be increased:Mild or mod hepatic failureFat malabsorption syndromesPatients on anti convulsants, glucocorticoids etc

Latent Autoimmune Diabetes in Latent Autoimmune Diabetes in AdultsAdults

IntroductionIntroductionDiabetes mellitusDiabetes mellitus ––

morbidity and premature deathmorbidity and premature death

Type 1 and 2 Type 1 and 2

Type 1 autoimmune process Type 1 autoimmune process –– destruction of pancreatic destruction of pancreatic insulin secreting beta cellsinsulin secreting beta cells

The antibodies The antibodies –– glutamicglutamic acid acid decarboxylasedecarboxylase and and insulinomainsulinoma associated antigen maybe found in bloodassociated antigen maybe found in blood

Type 2 often develops in adults Type 2 often develops in adults –– insuffinsuff insulin secretion insulin secretion with insulin resistancewith insulin resistance

UKPDS & LADAUKPDS & LADA10% of patients who appeared to have type 2 diabetes had antibod10% of patients who appeared to have type 2 diabetes had antibodies to ies to GAD and progressed to insulin dependencyGAD and progressed to insulin dependency

Among type 2 patients < 35 years the frequency is 25% Among type 2 patients < 35 years the frequency is 25%

These adults have a slowly developing type 1 diabetesThese adults have a slowly developing type 1 diabetes

They are initially non insulin requiring but progress to insulinThey are initially non insulin requiring but progress to insulin dependencydependency

The term latent autoimmune diabetes in adults is often applied tThe term latent autoimmune diabetes in adults is often applied to these o these patientspatients

Serum GAD acts as a marker for autoimmune damage associated withSerum GAD acts as a marker for autoimmune damage associated withLADALADA

At 10% prevalence this is a very common and important form of diAt 10% prevalence this is a very common and important form of diabetesabetes

Detecting patients with LADADetecting patients with LADA

Diagnosis is based on 3 criteria:Diagnosis is based on 3 criteria:Adult age at time of diabetesAdult age at time of diabetesPresence of circulating auto antibodies Presence of circulating auto antibodies Absence of a requirement for insulin at diagnosisAbsence of a requirement for insulin at diagnosis

These patients all have a stronger family history of both These patients all have a stronger family history of both type 1 and type 2 diabetes and will have a risk of other type 1 and type 2 diabetes and will have a risk of other autoimmune conditions similar to patients with classical autoimmune conditions similar to patients with classical type 1 diabetestype 1 diabetes

The extent of insulin resistance in LADA is debatable. The extent of insulin resistance in LADA is debatable.

Currently serum GAD antibody tests are not routinely Currently serum GAD antibody tests are not routinely requested, so patients with LADA will often be treated as requested, so patients with LADA will often be treated as if they have type 2 diabetes.if they have type 2 diabetes.

The correct diagnosis early in the disease might greatly The correct diagnosis early in the disease might greatly reduce the level of morbidity associated with the reduce the level of morbidity associated with the diabetes for patients with LADA. diabetes for patients with LADA.

However, the disease exists in the spectrum of However, the disease exists in the spectrum of autoimmune diabetes. autoimmune diabetes.

Diagnosis of LADADiagnosis of LADA

For diagnosis of type 1 diabetes, the presence of serum GAD antiFor diagnosis of type 1 diabetes, the presence of serum GAD antibodies has a bodies has a sensitivity of 75% and a specificity of100%.sensitivity of 75% and a specificity of100%.

The GAD antibody The GAD antibody titretitre gives a probability of the patientgives a probability of the patient’’s requirement for insulin. s requirement for insulin.

The higher the level, the faster and the more likely the progresThe higher the level, the faster and the more likely the progression to insulin sion to insulin dependence. dependence.

Plasma CPlasma C--peptide concentrations are used to assess pancreatic bpeptide concentrations are used to assess pancreatic b--cell failure as a cell failure as a marker of marker of endogeneousendogeneous insulin production.insulin production.

LADA can be ruled out using a random serum CLADA can be ruled out using a random serum C--peptide result, levels being peptide result, levels being significantly lower in the autoimmune diabetes patients. significantly lower in the autoimmune diabetes patients.

Fasting and stimulated plasma CFasting and stimulated plasma C--peptide levels have also been shown to be peptide levels have also been shown to be decreased in LADA patients.decreased in LADA patients.

It may be possible to use features of insulin deficiency such asIt may be possible to use features of insulin deficiency such as (relatively) high (relatively) high plasma glucose concentration with or without plasma glucose concentration with or without haemoglobinhaemoglobin (Hb)A1c greater than 10% (Hb)A1c greater than 10% despite good compliance to treatment, and lower body mass index despite good compliance to treatment, and lower body mass index (BMI), age and C(BMI), age and C--peptide level in order to identify patients most likely to have peptide level in order to identify patients most likely to have LADA.LADA.

Treatment of LADATreatment of LADALADA patients given SU drugs have poor fasting glucose LADA patients given SU drugs have poor fasting glucose

concentrations and higher levels of antibodies compared with thoconcentrations and higher levels of antibodies compared with those treated with se treated with insulin.insulin.

Type 2 patients who fail to respond to SU drugs are more likely Type 2 patients who fail to respond to SU drugs are more likely to be those with to be those with LADA.LADA.

Therefore, with the correct diagnosis, SU treatment could be avoTherefore, with the correct diagnosis, SU treatment could be avoided in LADA ided in LADA patients.patients.

There is an argument for the introduction of insulin very early There is an argument for the introduction of insulin very early in the disease process. in the disease process.

Treatment of patients with LADA is at an experimental stage, andTreatment of patients with LADA is at an experimental stage, and more work is more work is needed to determine whether insulin or needed to determine whether insulin or glitazonesglitazones (followed by insulin) or both is the (followed by insulin) or both is the best method.best method.

Therefore, it is not yet proven that early diagnosis leading to Therefore, it is not yet proven that early diagnosis leading to early initiation of insulin early initiation of insulin will alter clinical outcomes, but it is only through greater awawill alter clinical outcomes, but it is only through greater awareness and future reness and future research that this question can be answered research that this question can be answered

Recap: LADARecap: LADA

Describes patients with nonDescribes patients with non--insulin dependent diabetes who insulin dependent diabetes who progress to insulin dependency as their pancreatic secretion of progress to insulin dependency as their pancreatic secretion of insulin fails. insulin fails.

Diagnosis is based on adult age at the time of diabetes, the Diagnosis is based on adult age at the time of diabetes, the presence of serum presence of serum autoantibodiesautoantibodies to pancreatic antigens and the to pancreatic antigens and the absence of a requirement for insulin at diagnosis. absence of a requirement for insulin at diagnosis.

High High titrestitres of serum of serum glutamicglutamic acid acid decarboxylasedecarboxylase (GAD) antibodies (GAD) antibodies act as a marker for LADA.act as a marker for LADA.

Serum CSerum C--peptide concentrations are also lower in autoimmune peptide concentrations are also lower in autoimmune diabetic patients. diabetic patients.

The best treatment for patients with LADA is not clear, but earlThe best treatment for patients with LADA is not clear, but early y insulin treatment may prevent pancreatic binsulin treatment may prevent pancreatic b--cell failure cell failure

CASESCASES

Patient ID Patient ID 45 year old45 year old WomanWoman

Clinical Notes on Request Form :Clinical Notes on Request Form :Past history of gestational diabetesPast history of gestational diabetes..

Case DetailsCase Details75g glucose tolerance test75g glucose tolerance test

0 minute glucose 0 minute glucose 5.8 mmol/L 5.8 mmol/L 60 minute glucose 60 minute glucose 13.1 mmol/L 13.1 mmol/L 120 minute glucose 5.6 mmol/L120 minute glucose 5.6 mmol/L

Additional Information:Additional Information:Previous fasting glucose ofPrevious fasting glucose of 6.1 6.1 mmolmmol/L /L

Suggested CommentSuggested CommentNormal glucose tolerance test. Normal glucose tolerance test.

Suggest repeat fasting glucose in 12 months time.Suggest repeat fasting glucose in 12 months time.

The three main questions on this case are The three main questions on this case are the diagnosis, the diagnosis,

the tool to use for followthe tool to use for follow--up (fasting glucose or up (fasting glucose or OGTT) and OGTT) and

the timing of any followthe timing of any follow--upup. .

Patient ID : Patient ID : 92 year old woman Patient92 year old woman Patient

Location : Location : Nursing HomeNursing Home

Clinical Notes on Request FormClinical Notes on Request Form””?Temporal ?Temporal arteritisarteritis. Increased ESR.. Increased ESR.

Case Details:Case Details:Serum Serum carbamazepinecarbamazepine level: <3 level: <3 umolumol/L /L (Therapeutic Range 13(Therapeutic Range 13--38 38 umolumol/L)/L)

Additional Information:Additional Information:List of medications:List of medications:isosorbideisosorbide, , diltiazemdiltiazem, aspirin, hydrochlorothiazide, , aspirin, hydrochlorothiazide, thyroxinethyroxine, , neocytamenneocytamen, , caltratecaltrate, , ostelinostelin, , macuvisionmacuvision, , loseclosec and prednisone.and prednisone.

Suggested CommentSuggested CommentUndetectable Undetectable carbamazepinecarbamazepine. . Suggest check authenticity of request and Suggest check authenticity of request and compliance.compliance.

One might consider interaction with One might consider interaction with cytochromecytochromeP4503A4 (CYP 3A4) inducing agents, leading to a P4503A4 (CYP 3A4) inducing agents, leading to a reduced concentration although none of the other drugs reduced concentration although none of the other drugs

listed falls into this category.listed falls into this category.

On the contrary, On the contrary, diltiazemdiltiazem is an inhibitor of CYP 3A4 and is an inhibitor of CYP 3A4 and might be expected to increase might be expected to increase carbamazepinecarbamazepine levels.levels.

Patient ID : Patient ID : 33 year old female Patient33 year old female Patient

Clinical Notes on Request FormClinical Notes on Request FormNonNon--alcoholic alcoholic steatohepatitissteatohepatitis (NASH) for a number (NASH) for a number of of years. years. BMIBMI = 38= 38

Case DetailsCase DetailsCholesterol 7.4 Cholesterol 7.4 mmolmmol/L/LTriglycerides 2.0 Triglycerides 2.0 mmolmmol/L/LLDLLDL--C 4.7 C 4.7 mmolmmol/L/LHDLHDL--C 1.8 C 1.8 mmolmmol/L/L

Additional InformationAdditional InformationNot on any medication.Not on any medication.

Raised total and LDLRaised total and LDL--cholesterol and a history of NASH cholesterol and a history of NASH are both associated with increased risk of cardiovascular are both associated with increased risk of cardiovascular

diseasedisease..

Exclude secondary causes of Exclude secondary causes of hypercholesterolaemiahypercholesterolaemia and and do full cardiovascular risk assessment.do full cardiovascular risk assessment.

Lifestyle interventions to increase exercise and promote Lifestyle interventions to increase exercise and promote

weight loss are recommendedweight loss are recommended..

NonNon--alcoholic fatty liver disease (NAFLD) encompasses a spectrum of alcoholic fatty liver disease (NAFLD) encompasses a spectrum of liver liver disease from disease from steatosissteatosis to NASH, cirrhosis and rarely to NASH, cirrhosis and rarely hepatocellularhepatocellularcarcinoma. carcinoma.

Prospective studies have shown that survival with NASH is reduceProspective studies have shown that survival with NASH is reduced from d from cardiovascular and liver related causes.cardiovascular and liver related causes.

NAFLD is strongly associated with obesity and insulin resistancNAFLD is strongly associated with obesity and insulin resistance and other e and other features of the metabolic syndrome. features of the metabolic syndrome.

This is a case of a severely obese lady with documented NASH andThis is a case of a severely obese lady with documented NASH and lipid lipid studies showing raised total and LDLstudies showing raised total and LDL--cholesterol. cholesterol.

The lipid results are not typical of the metabolic syndrome in tThe lipid results are not typical of the metabolic syndrome in this case his case making it important to exclude treatable secondary causes in addmaking it important to exclude treatable secondary causes in addition to ition to doing a full cardiovascular risk assessment. doing a full cardiovascular risk assessment.

Although insulin resistance is central to NAFLD, routine insulinAlthough insulin resistance is central to NAFLD, routine insulinmeasurement is not helpful. measurement is not helpful.

Regression of histological features of NASH has been Regression of histological features of NASH has been demostrateddemostrated with with weight loss. weight loss. Reference: Riley P, Reference: Riley P, O'DonhoueO'Donhoue J, Crook M. A growing burden: the pathogenesis, investigation aJ, Crook M. A growing burden: the pathogenesis, investigation and management of nonnd management of non--alcholicalcholicfatty liver disease. J fatty liver disease. J ClinClin PatholPathol. 2007 May 4; [. 2007 May 4; [EpubEpub ahead of print]ahead of print]

Patient ID : Patient ID : 31 year old female Patient31 year old female Patient

Location Location MOPDMOPD

Clinical Notes on Request Form:Clinical Notes on Request Form:Feeling tired and lethargic.Feeling tired and lethargic.

Case DetailsCase DetailsFT4 20FT4 20 pmolpmol/L /L (9(9--19) 19) TSH 0.02 TSH 0.02 mUmU/L /L (0.4(0.4--4.0)4.0)FT3 5.5 FT3 5.5 pmolpmol/L /L (3.0(3.0--5.5)5.5)

Additional InformationAdditional InformationNot on any medication.Not on any medication.

The combination of a suppressed TSH (<0.1mU/L) and The combination of a suppressed TSH (<0.1mU/L) and borderline/raised fT3 and fT4 concentrations suggests borderline/raised fT3 and fT4 concentrations suggests hyperthyroidism (due to Graves' disease, hyperthyroidism (due to Graves' disease, thyroiditisthyroiditis, or surreptitious , or surreptitious T4 intake). T4 intake).

TSHTSH--receptor receptor AbAb and thyroid uptake scan may be indicated. and thyroid uptake scan may be indicated.

However in a woman of childbearing age, However in a woman of childbearing age, pregancypregancy should be should be considered as TSH may be low in the first trimester of pregnancyconsidered as TSH may be low in the first trimester of pregnancy, , although such extremes of TSH and fT4 (in response to a although such extremes of TSH and fT4 (in response to a significantly increased significantly increased hCGhCG) are usually seen in the ) are usually seen in the hyperemesishyperemesisgroup. group.

If she was found to be pregnant, the tests should be repeated inIf she was found to be pregnant, the tests should be repeated in 6 6 weeks time. weeks time.

If fT4 and fT3 were clearly elevated as opposed to borderline inIf fT4 and fT3 were clearly elevated as opposed to borderline in a a pregnant woman, then the diagnosis would be gestational pregnant woman, then the diagnosis would be gestational thyrotoxicosisthyrotoxicosis and would warrant treatment.and would warrant treatment.