CARBOHYRATES IN CARBOHYRATES IN CLINICAL BIOCHEMISTRYCLINICAL BIOCHEMISTRY

ProfProf.. Dr. Serdar Öztezcan Dr. Serdar Öztezcan

Regulation of blood Regulation of blood glucose concentration glucose concentration

The concentration of glucose in the The concentration of glucose in the blood is regulated by a complex blood is regulated by a complex interplay of interplay of multiple pathwaysmultiple pathways– GlycogenesisGlycogenesis– GlycogenolysisGlycogenolysis– GlyconeogenesisGlyconeogenesis

HormonesHormones

The concentration of glucose in the blood The concentration of glucose in the blood is normally maintained within a narrow is normally maintained within a narrow interval by hormones :interval by hormones :– Insulin Insulin – Counterregulatory hormones Counterregulatory hormones

• GlucagonGlucagon• EpinephrineEpinephrine• CortisolCortisol• Growth hormoneGrowth hormone

InsulinInsulin

Anabolic hormone Anabolic hormone – stimulates the uptake of glucose into fat stimulates the uptake of glucose into fat

and muscleand muscle– promotes the conversion of glucose to promotes the conversion of glucose to

glycogen or fat for storageglycogen or fat for storage– inhibits glucose production by the liverinhibits glucose production by the liver– stimulates protein synthesis and inhibits stimulates protein synthesis and inhibits

protein breakdownprotein breakdown– uptake ions (specially Kuptake ions (specially K++ and PO and PO44

-3-3))

The major insulin target organs are the The major insulin target organs are the liver, liver, skeletal muscle skeletal muscle and and adipose tissueadipose tissue

InsulinInsulin

Counterregulatory Counterregulatory HormonesHormones These hormones are These hormones are catabolic catabolic

– increase hepatic glucose productionincrease hepatic glucose production•initially by enhancing the initially by enhancing the

breakdown of glycogen to glucose breakdown of glycogen to glucose (glycogenolysis) (glycogenolysis)

•later by stimulating the synthesis of later by stimulating the synthesis of glucose glucose (glyconeogenesis)(glyconeogenesis)

Diabetes Mellitus Diabetes Mellitus Dm is actually a group of metabolic diseases Dm is actually a group of metabolic diseases

characterized by characterized by hyperglycemiahyperglycemia resulting from resulting from defects in insulindefects in insulin secretion, secretion, insulininsulin action,action, oror bothboth

Clasification and diagnostic scheme for diabetes Clasification and diagnostic scheme for diabetes mellitus (ADA/WHO)mellitus (ADA/WHO)– Type 1 diabetesType 1 diabetes– Type 2 diabetesType 2 diabetes– Other specific types of diabetesOther specific types of diabetes– Gestational diabetes mellitusGestational diabetes mellitus

– Impaired Fasting Glucose Impaired Fasting Glucose – Impaired Glucose ToleranceImpaired Glucose Tolerance

Type 1 diabetesType 1 diabetes Formerly known as IDDM or juvenile-onset Formerly known as IDDM or juvenile-onset

diabetesdiabetes Approximatelly Approximatelly 5-10% 5-10% of all individuals with of all individuals with

diabetes mellitus are in this categorydiabetes mellitus are in this category Symptoms (eg. Polyuria, polydipsia, and rapid Symptoms (eg. Polyuria, polydipsia, and rapid

weight loss) usually present weight loss) usually present acutelyacutely Patients has Patients has insulinopenia insulinopenia because of loss of because of loss of

pancreatic islet pancreatic islet cells cells Depend on Depend on insulin treatment insulin treatment to sustain life and to sustain life and

prevent ketosisprevent ketosis Most individuals have antibodies that identify Most individuals have antibodies that identify

an autoimmun prosessan autoimmun prosess

Type 2 diabetesType 2 diabetes Formerly known as NIDDM Formerly known as NIDDM This type consititues approx. This type consititues approx. 90% 90% of all cases of all cases

Patients have minimal symptoms and are not Patients have minimal symptoms and are not prone to ketosisprone to ketosis

Insulin concentration may be Insulin concentration may be within the within the reference interval, decreased, or increased reference interval, decreased, or increased

Most people with this form of diabetes have Most people with this form of diabetes have impaired insulin actionimpaired insulin action

ObesityObesity is commonly associated, and weight is commonly associated, and weight loss alone usually improves the hyperglycemialoss alone usually improves the hyperglycemia

Many individuals may require Many individuals may require dietary dietary manipulationmanipulation, an , an oral hypoglycemicoral hypoglycemic agent, or agent, or insulin insulin terapyterapy

Other specific types of Other specific types of diabetesdiabetes This subclass is associated This subclass is associated with certain conditions with certain conditions

such as such as – Genetic defects of Genetic defects of -cell function-cell function– Genetic defects in insulin actionGenetic defects in insulin action– Disease of exocrine pancreasDisease of exocrine pancreas– Endocrinopathies (e.g., Cushing disease, Endocrinopathies (e.g., Cushing disease,

acromegaly, glucagonoma)acromegaly, glucagonoma)– The administration of hormones and drugs known The administration of hormones and drugs known

to to induce induce -cell dysfunction -cell dysfunction (e.g., dilantine and (e.g., dilantine and pentamidine) or pentamidine) or impair insulin action impair insulin action (e.g., (e.g., glucocorticoides, thiazides, and glucocorticoides, thiazides, and -adrenergics)-adrenergics)

– InfectionsInfections The characteristics and prognosis of this form of The characteristics and prognosis of this form of

diabetes diabetes depends on the primary disorderdepends on the primary disorder

Gestational diabetes Gestational diabetes mellitusmellitus GDM is “any degree of carbohyrates GDM is “any degree of carbohyrates

intolerance with intolerance with onset or first onset or first recognation recognation during pregnancy”during pregnancy”

Patient with GDM frequently return to Patient with GDM frequently return to normal postpartumnormal postpartum

However GDM is associated with However GDM is associated with increased perinatal complications and increased perinatal complications and an increase risk for development of an increase risk for development of diabets diabets (predominantly Type 2) in later (predominantly Type 2) in later yearsyears

Impaired Fasting Glucose Impaired Fasting Glucose and Impaired Glucose and Impaired Glucose ToleranceTolerance

Impaired fasting glucoseImpaired fasting glucose– is diagnosed in people who have a is diagnosed in people who have a

fasting glucose value above normal but fasting glucose value above normal but below below the concentration for diagnosis the concentration for diagnosis of of diabetesdiabetes

– It is a metabolic stage between normal It is a metabolic stage between normal glucose homeostasis and diabetesglucose homeostasis and diabetes

Impaired Fasting Glucose Impaired Fasting Glucose and Impaired Glucose and Impaired Glucose ToleranceTolerance Impaired glucose toleranceImpaired glucose tolerance

– OGTT is required to assign a patient OGTT is required to assign a patient to this classto this class

– have plasma glucose response have plasma glucose response between normal and diabetic states between normal and diabetic states during the OGTTduring the OGTT

– Development of overt diabetes occurs Development of overt diabetes occurs at a rate 1-5% per year in people with at a rate 1-5% per year in people with IGTIGT

Complications of DMComplications of DM

Diabetic ketoacidosis Diabetic ketoacidosis – arises from a number of metabolic problems arises from a number of metabolic problems

caused by insulin lackcaused by insulin lackHyperosmolar non-ketotic coma Hyperosmolar non-ketotic coma

(HONK)(HONK)– occurs mostly in elderly, tip 2 diabeticsoccurs mostly in elderly, tip 2 diabetics– level of insulin is sufficient to prevent ketosis level of insulin is sufficient to prevent ketosis

but does not prevent hyperglycemia and but does not prevent hyperglycemia and osmotic diuresisosmotic diuresis

Late complications of DMLate complications of DM

DM is not only characterized by the DM is not only characterized by the presence of hyperglycaemia but also by presence of hyperglycaemia but also by the occurrence of late complicationsthe occurrence of late complications – Microangiopathy Microangiopathy

• RetionopathyRetionopathy• NephropathyNephropathy• NeuropathyNeuropathy

– MacroangiopathyMacroangiopathy• AteroscleorosisAteroscleorosis

Diagnosis of diabetes Diagnosis of diabetes mellitusmellitus Diagnosis depends solely on the Diagnosis depends solely on the

demonstration of hyperglycemia demonstration of hyperglycemia – Diagnosis of type 1 is usually Diagnosis of type 1 is usually easyeasy because because

• hyperglycemia appears abruptly, severe, hyperglycemia appears abruptly, severe, accompained by serious metabolic problemsaccompained by serious metabolic problems

– Diagnosis of type 2 may be Diagnosis of type 2 may be difficultdifficult because because• the metabolic changes are often not severe the metabolic changes are often not severe

enough for the patient to notice symptomsenough for the patient to notice symptoms• Complications are Complications are present in approximately present in approximately

30% of patients at clinical diagnosis of type 2 30% of patients at clinical diagnosis of type 2 diabetsdiabets

Diagnosis of diabetes Diagnosis of diabetes mellitusmellitus

All adults All adults older than age 45 years older than age 45 years should have a should have a measurement of fasting blood glucose measurement of fasting blood glucose every 3 yearsevery 3 years

Earlier age or more frequently Earlier age or more frequently in individuals who in individuals who display;display;– Obesity (BMI of 27 kg/mObesity (BMI of 27 kg/m22))– Family history of diabetes in a first-degree Family history of diabetes in a first-degree

relativerelative– History of GDM or delivering a baby > 4.1 kgHistory of GDM or delivering a baby > 4.1 kg– Hypertension (> 140/90 mm Hg)Hypertension (> 140/90 mm Hg)– Low HDL concentration (< 35mg/dL)Low HDL concentration (< 35mg/dL)– Elevated triglyceride concentrations (> 250 Elevated triglyceride concentrations (> 250

mg/dL)mg/dL)– A history of IGF or OGTA history of IGF or OGT

Criteria for the diagnosis Criteria for the diagnosis of diabetes mellitusof diabetes mellitus

Any one of the following methods is diagnostic;Any one of the following methods is diagnostic;

– Classic Classic symptomssymptoms of diabetes + of diabetes + causal/random plasma glucose causal/random plasma glucose concentration concentration > 200 mg/dL> 200 mg/dL

– FastingFasting plasma glucose plasma glucose > 126 mg/dL> 126 mg/dL

– Two-hourTwo-hour plasma glucose plasma glucose > 200 mg/dL> 200 mg/dL during the during the OGTTOGTT

* If, any three criteria is positive, it must be confirmed on a * If, any three criteria is positive, it must be confirmed on a subsequent day by any of the three methodssubsequent day by any of the three methods

Criteria for the diagnosis Criteria for the diagnosis of diabetes mellitusof diabetes mellitus Intermediate groupIntermediate group

– Impaired Fasting GlucoseImpaired Fasting Glucose•Fasting plasma glucose between Fasting plasma glucose between 100 100

(106) and 125 mg/dL.(106) and 125 mg/dL.

– Impaired Glucose ToleranceImpaired Glucose Tolerance•Fasting plasma glucose Fasting plasma glucose < 126 mg/dL< 126 mg/dL•Two-hour OGTT plasma glucose Two-hour OGTT plasma glucose

concentration is between concentration is between 140 and 140 and 199 mg/dL199 mg/dL

Oral glucose tolerance Oral glucose tolerance test (OGTT)test (OGTT)

In OGTT plasma glucose concentrations In OGTT plasma glucose concentrations are measured fasting, then every 30 are measured fasting, then every 30 minutes for 2 hours after an oral minutes for 2 hours after an oral glucose loadglucose load

Although Although more sensitive than FPG more sensitive than FPG determinations, OGTT is affected by a determinations, OGTT is affected by a large number of factors that result in large number of factors that result in poor reproducibilitypoor reproducibility

Oral glucose tolerance Oral glucose tolerance test (OGTT)test (OGTT)

The following conditions should be met for The following conditions should be met for performing an OGTTperforming an OGTT;;– discontinue, when possible, medications known discontinue, when possible, medications known

to affect it (thiazides, oc, corticosteroids)to affect it (thiazides, oc, corticosteroids)– in the morning after 3 days of unrestricted diet in the morning after 3 days of unrestricted diet

(150 g ch/day) and activity(150 g ch/day) and activity– after a 10-14 hour fastafter a 10-14 hour fast– seated during the test without smoking seated during the test without smoking

cigarettes and drinking coffee ect.cigarettes and drinking coffee ect.– For nonpregnant adults 75 g, For nonpregnant adults 75 g, – for children 1.75 g/kg (max 75g ),for children 1.75 g/kg (max 75g ),– dissolved in 300 ml water / 5 minutesdissolved in 300 ml water / 5 minutes

Diagnosis of GDMDiagnosis of GDM

ADA recommentation for ADA recommentation for laboratory diagnosis of GDM arelaboratory diagnosis of GDM are– Low-risk Low-risk patients require no testingpatients require no testing– Average-risk Average-risk patients should be patients should be

testing at testing at 24 to 28 weeks of gestation24 to 28 weeks of gestation– High-riskHigh-risk patients should undergo patients should undergo

immediate testingimmediate testing

Diagnosis of GDMDiagnosis of GDM

Risk factors for GDM include,Risk factors for GDM include,– Family history of DM in a first-degree Family history of DM in a first-degree

relativerelative– ObesityObesity– Advanced maternal ageAdvanced maternal age– GlycosuriaGlycosuria– History of poor obstetric outcome (stillbirth History of poor obstetric outcome (stillbirth

or macrosomia)or macrosomia)– Member of an ethnic group with a high Member of an ethnic group with a high

prevalence of GDMprevalence of GDM

Diagnosis of GDMDiagnosis of GDM

First step is identical to that for First step is identical to that for diagnose diabetes in a nonpregnant diagnose diabetes in a nonpregnant individual individual (Fasting >126 or random (Fasting >126 or random >200)>200)

However, in the absence of that degree However, in the absence of that degree of hyperglycemia, average and high risk of hyperglycemia, average and high risk patients patients receivereceive a glucose tolerance a glucose tolerance test following one of two methods;test following one of two methods;– One step testOne step test– Two step testTwo step test

Diagnosis of GDMDiagnosis of GDM

One step test One step test ; perform a ; perform a 100 g OGTT for 3 100 g OGTT for 3 hourhour (or 75 g for 2 hour - it is not as well (or 75 g for 2 hour - it is not as well validated as the 100 g test)validated as the 100 g test)

Two step test Two step test ; the first step is a ; the first step is a 50 g50 g oral oral glucose load (the patient does not need to glucose load (the patient does not need to be fasting) followed by a plasma glucose be fasting) followed by a plasma glucose determination at determination at 1 hour1 hour– A value grater than or equal to A value grater than or equal to 140 mg /dL 140 mg /dL

(or 130)(or 130) indicates the necessity for definive indicates the necessity for definive testing (one step test)testing (one step test)• 90%-130, 80% 14090%-130, 80% 140

Diagnosis of GDMDiagnosis of GDM

To diagnose GDM with 100 g test at To diagnose GDM with 100 g test at least least two valuestwo values must meet or exceed must meet or exceed the following,the following,– FastingFasting 95mg/dL95mg/dL– 1 hr1 hr 180mg/dL180mg/dL – 2 hr2 hr 155mg/dL155mg/dL– 3 hr3 hr 140mg/dL140mg/dL

Gestational diabetes Gestational diabetes mellitusmellitus

At 6 to 12 weeks postpartumAt 6 to 12 weeks postpartum, all , all patients who had GMD should be patients who had GMD should be evaluated for diabetes evaluated for diabetes

if not present, be reevaluated for if not present, be reevaluated for diabetes diabetes at least every 3 yearsat least every 3 years

Infants born to mothers with Infants born to mothers with diabetesdiabetes Infants born to mothers with diabetes are Infants born to mothers with diabetes are

increased risk for increased risk for respiratuary distress respiratuary distress syndrome, hypocalcemia and, syndrome, hypocalcemia and, hyperbilirubinemiahyperbilirubinemia

Fetal insulin secretion is stimulated in the Fetal insulin secretion is stimulated in the neonate of mother with diabetes. When the neonate of mother with diabetes. When the infant is born and the umblical cord is severed, infant is born and the umblical cord is severed, the infant’s oversupply of glucose is abruptly the infant’s oversupply of glucose is abruptly terminated, terminated, causing severe hypoglycemiacausing severe hypoglycemia

HypoglycemiaHypoglycemia

Hypoglycemia involves decreased Hypoglycemia involves decreased plasma glucose levels and can have plasma glucose levels and can have many causesmany causes– Some are Some are transient and relatively transient and relatively

insignificantinsignificant– Others can be Others can be life threating life threating

Glucagon and other glycemic factors Glucagon and other glycemic factors are released is betweenare released is between 65-70 mg/dL65-70 mg/dL

Symptoms of hypoglycemia appear at Symptoms of hypoglycemia appear at about about 50-55 mg/dL50-55 mg/dL

HypoglycemiaHypoglycemia

Hypoglycemia was classified asHypoglycemia was classified as– postabsorbative (fasting) postabsorbative (fasting) – postprandial (reactive)postprandial (reactive)

Current approaches suggest Current approaches suggest classification based on clinical classification based on clinical characteristics. This classification characteristics. This classification separates patients into those who separates patients into those who appearappear healthyhealthy oror sicksick

HypoglycemiaHypoglycemia

Symptoms of hypoglycemia may be Symptoms of hypoglycemia may be categorized as neurogenic categorized as neurogenic (adrenergic) or neuroglycopenic(adrenergic) or neuroglycopenic– NeurogenicNeurogenic symptoms include sweating, symptoms include sweating,

shakiness, tachycardia, and anxietshakiness, tachycardia, and anxietyy– NeuroglycopenicNeuroglycopenic symptoms include symptoms include

weakness, tiredness, or dizziness; weakness, tiredness, or dizziness; difficulty with concentration; confusion; difficulty with concentration; confusion; blurred vision; and, in extreme casesblurred vision; and, in extreme cases, , coma and deathcoma and death

HypoglycemiaHypoglycemia

Some of causes of hypoglycemiaSome of causes of hypoglycemia– Drugs (Drugs (insulininsulin, oral ) and ethanol, oral ) and ethanol– InsulinomaInsulinoma– Severe exerciseSevere exercise– Glucogen storage diseases:Glucogen storage diseases:

• Glucose 6-phospathase deficiency tip 1 (von Gierke)Glucose 6-phospathase deficiency tip 1 (von Gierke)– Neonatal hypoglycemiaNeonatal hypoglycemia– Galactosemia Galactosemia – Hormonal deficiencies - Hormonal deficiencies - ccortisol, growth hormone (in ortisol, growth hormone (in

children), glucagon, and epinephrinechildren), glucagon, and epinephrine– Critical illnesses - Critical illnesses - ccardiac, hepatic, and renal diseasesardiac, hepatic, and renal diseases, ,

sepsissepsis– Congenital - Congenital - ccarnitine deficiencyarnitine deficiency

Role of laboratoryRole of laboratory

Glucose measurementGlucose measurement– Serum or plasma glucose concentrationSerum or plasma glucose concentration– Self-monitoring of whole blood glucose Self-monitoring of whole blood glucose

concentrations (capillary)concentrations (capillary)– Urine ( Treshold 170-180 mg/dL )Urine ( Treshold 170-180 mg/dL )

Glucose tolerance testsGlucose tolerance tests Glycosylated hemoglobine Glycosylated hemoglobine KetonesKetones MicroalbuminuriaMicroalbuminuria Islet autoantibodyIslet autoantibody InsulinInsulin

Glycosylated hemoglobineGlycosylated hemoglobine

Long term blood glucose regulation can be Long term blood glucose regulation can be followed by measurement of glycosylated followed by measurement of glycosylated hemoglobine hemoglobine

Formation of a hemoglobin compound when Formation of a hemoglobin compound when glucose reacts with the amino group of glucose reacts with the amino group of hemoglobinhemoglobin

The rate of formation is directly proportional to The rate of formation is directly proportional to the plasma glucose concentrationsthe plasma glucose concentrations

Reflects the avarage blood glucose level over Reflects the avarage blood glucose level over the the previous 2-3 monthsprevious 2-3 months

Hemoglobin A1c the most commonHemoglobin A1c the most common Reference values Reference values 4,5 to 6% (> 6,5 diagnose ?)4,5 to 6% (> 6,5 diagnose ?) %1 --- 35mg/dL%1 --- 35mg/dL

FructosamineFructosamine

Refers to the ketoamin linkage between Refers to the ketoamin linkage between glucose and proteinglucose and protein

Since all serum protein can be glycated Since all serum protein can be glycated and albumin is the most abundant protein and albumin is the most abundant protein in serum, measurement of fructosamin is in serum, measurement of fructosamin is largely a measurement of largely a measurement of albuminalbumin

Measure of glycemic control during the 3-Measure of glycemic control during the 3-week period before sampling, since the week period before sampling, since the half-life of albumin is half-life of albumin is 2 to 3 weeks2 to 3 weeks

KetonesKetones

Ketone bodies are produced by the liver through Ketone bodies are produced by the liver through metabolism of metabolism of fatty acid fatty acid to provide a ready energy to provide a ready energy source from stored lipids at times of source from stored lipids at times of low carbohyrate low carbohyrate availability availability such assuch as– diabetes mellitus, starvation/fasting, high-fat diets, diabetes mellitus, starvation/fasting, high-fat diets,

prolonged vomiting, and glycogen storage diseaseprolonged vomiting, and glycogen storage disease The three ketone bodies areThe three ketone bodies are

– aceton (2%),aceton (2%),– acetoacetic acid (20%),acetoacetic acid (20%),– -hidroxybutyric acid (78%)-hidroxybutyric acid (78%)

Recommended for patients with type 1 diabetes Recommended for patients with type 1 diabetes duringduring– acute illness, stress, pregnancy, eleveted blood acute illness, stress, pregnancy, eleveted blood

glucose above 300 mg/dL or when patient has glucose above 300 mg/dL or when patient has sings of ketoacidosissings of ketoacidosis

MicroalbuminuriaMicroalbuminuria

Diabetes mellitus causes progressive changes Diabetes mellitus causes progressive changes to the kidneys and ultimately results in to the kidneys and ultimately results in diabetic nephropathydiabetic nephropathy

This complication progesses over years and This complication progesses over years and may be delayed by aggresive glycemic controlmay be delayed by aggresive glycemic control

An early sign is an An early sign is an increase in urinary increase in urinary albuminealbumine

Microalbumin measurements are useful to Microalbumin measurements are useful to assist in diagnosis assist in diagnosis at an early stage at an early stage andand before before the development of proteinuriathe development of proteinuria

Microalbumin Microalbumin 30-300 mg/day30-300 mg/day (reference (reference interval <30 mg/day)interval <30 mg/day)

Proteinuria; greater than Proteinuria; greater than 0,3/0,5 g/day0,3/0,5 g/day

Islet autoantibody and Islet autoantibody and insulininsulin Islet autoantibodyIslet autoantibody

– The presence of autoantibodies to the The presence of autoantibodies to the cells of the cells of the pancreas is charactheristic of type 1 pancreas is charactheristic of type 1

– Testing is not currently recommended for rutine Testing is not currently recommended for rutine screening for diabetes diagnosis screening for diabetes diagnosis

– In the future, it might identify at-risk, prediabetik In the future, it might identify at-risk, prediabetik patientspatients

InsulinInsulin– Not required for the diagnosis of diabetes mellitusNot required for the diagnosis of diabetes mellitus– Insulin resistanceInsulin resistance– However, ın certain However, ın certain hypoglycemic stateshypoglycemic states, it is , it is

important to know the concentration of insulin in important to know the concentration of insulin in relation to the plasma glucose concentrationrelation to the plasma glucose concentration

-insulinoma-insulinoma

HOMA-IRHOMA-IR

The homeostatic model assessment The homeostatic model assessment (HOMA) is a method used to quantify (HOMA) is a method used to quantify insulin resistance and beta-cellinsulin resistance and beta-cell functionfunction

It was first described under the name It was first described under the name HOMA by Matthews HOMA by Matthews et al.et al. in 1985 in 1985

Glucose X Insulin X 0,055Glucose X Insulin X 0,055 : <2.3 : <2.3 22.522.5

Case studyCase study Fasting glucose 95Fasting glucose 95 Fasting glucose 135Fasting glucose 135 Fasting glucose 115Fasting glucose 115 Random glucose 202Random glucose 202 OGTT 2 hour 145OGTT 2 hour 145 OGTT 2 hour 207OGTT 2 hour 207 Serial measurementSerial measurement

– 1. quarter 1. quarter HbA1c 7.8% HbA1c 7.8% FPG 280FPG 280– 2. quarter 2. quarter HbA1c 15.3% HbA1c 15.3% FPG 85FPG 85– 3. quarter 3. quarter HbA1c 8.5% HbA1c 8.5% FPG 91FPG 91

Two testTwo test– Self monitoring 200Self monitoring 200– In the laboratory with serumIn the laboratory with serum