Clinical Biochemistry and Preanalytical Variations
Laboratory Medicine: is an integral component of patient care. 60% to 70% of medical decisions are based
laboratory medicine .Lab tests consume about 2.3% of annual health care costs in the United States.
Laboratory results enable healthcare providers to: Assess early disease risk. Opt for preventive therapies or less-
invasive treatment. Select and monitor appropriate treatment. Follow the natural progression of diseases.
Laboratory medicine means: The discipline involved in the Selection, Provision,Interpretation of diagnostic testing
that uses primarily samples from patients.
Laboratory medicine is comprised of: pathologists, lab scientists, technicians and technologist.
Labs are directed by clinical pathologists, specialists have a broad range of knowledge regarding laboratory testing
and many are further subspecialized. Clinical pathologists are available for consultation.
The ‘Brain-to-Brain’ loop, depicting the steps in the process of considering, performing and using laboratory tests for
diagnosis. İt goes from laboratorians brain to physician’s brain to patient.
Diagnosting testins phases: Preanalytic phase, Analytic phase ,Post analytic phase.
Preanalytic phase: Test Order, Patient Preparation, Sample Collection, Sample Transport, Sample Process. % 50 to
70 errors occur in this phase.
Test ordering: Laboratory tests can be used for multiple clinical purposes: Screening, Risk assessment ,Establishment
of a diagnosis ,Support of a diagnosis ,Exclusion of a diagnosis ,Prognosis, Determination of individualized therapy
,Assessment of disease progression or response to therapy.
Specimen Collection and Processing: Proper collection, processing, storage, and transport of common sample types
associated with requests for diagnostic testing are critical to the provision of quality test results.
Identification: Proper identification of the patient is essential, and the sample should be properly labeled at all steps
if separated from the primary collection container.
The minimum information on any label associated with a specific specimen should include :
 Patient’s name
 Location
 Identifying number
 Date and time of collection
Type of specimens: Blood,Whole blood, serum, plasma, urine, feces, saliva, spinal, synovial, amniotic.
Blood samples: Are obtained from veins, arteries, capillaries.
Venous blood is the specimen of choice. Arterial in blood gas analyses, capillaries in children and POCT.
Arter ve ven kan örnekleri arasında farklılıklar gözlenir : glukoz ve oksijen arter kanında yüksekken, laktat ve
amonyak düzeyleri venöz kanda daha yüksektir.
Blood collection tubes:There are a lot of types of tubs, they vary by presence or absence and type of additive, and
volume of tube. Different types of additives are identified by the color of the stopper thats used.
Color: Tube/Used for.
 Blue: Sodium citrate/Coagulation,PT,PTT.
 Red: No additive or clot activator/ serology,drugs,SPEP and CHEM.
 Yellow: No additive or clot activator + gel / Serology, drugs, SPEP and CHEM.
 Dark blue: Special tube and stopper with clot activator/Trace and some toxicology.
 Light green: Lithium heparin/ Most Chem and cytogenetics
 Dark green: Sodium heparin/Most Chem and cytogenetics.
 Purple:Potassium EDTA/ HEME,PCR,quants,virüs,DNA ammonia and ACTH
 Gray: Sodium flüoride or oxalate/Glucose, volatiles,lactate,and autopsies.
 Yellow: Acid citrate dextrose/ Molecular diagnostics and cytology.
Anticoagulants and Preservatives for Blood:
 Heparin: Amonyak, kan gazları, methemoglobin analizi
 Ethylenediaminetetraacetic Acid (EDTA): is a chelating agent of divalent cations such as Ca2+ and Mg2+
 Sodium Fluoride
 Citrate
 Acid Citrate Dextrose
Edtanın anticoagulan kullanımında: Azalan testler: Ca+2, Mg+2, Amilaz,Alkalen fosfataz, PTH, Renin, Aldosteron.
Artan Testler: K+ veya Na+ (Edta tuzuna göre)
Serum is defined as the liquid portion of blood that remains after coagulation has occurred. Most common.
Plasma is defined as the non-cellular component of anticoagulated whole blood.
Venipuncture: Doğru antikoagülan kullanımının kontrolü, En az venöz staz ile kan alınması biyolojik tehlikelerin
belirtilmesi.
Handling the specimens for analysis: Identification,Preservation,Separation and storage ,Transport.
Preservation: For some tests, specimens must be kept at 4°C from the time the blood is drawn until the specimens
are analyzed or until the serum or plasma is separated from the cells. Example: Ammonia, Blood gas determinations
(PCO2, PO2, and blood pH, etc.)
Separation and Storage of Specimens: Plasma or serum should be separated from cells as soon as possible and
certainly within 2 hours for some but not all analytes. If it is impossible to centrifuge a blood specimen within 2
hours, the specimen should be held at room temperature. For most plasma samples used for molecular diagnostics,
the plasma should be removed from the primary tube promptly a er centrifuga on and held at −20°C in a freezer
capable of maintaining this temperature
Transport: After collection, specimens should be transported to a laboratory, processed, and analyzed as soon as
possible as delays can compromise the results. Glucose, for instance, decreases at a rate of 5%-7% per hour in whole
blood atroom temperature.
Urine samples: The type of urine specimen to be collected is dictated by the tests to be performed. Untimed
(Random) , Timed (Predetermined interval of time) 4, 12, or 24 hours -> Volume must be recorded.
A clean, early morning, fasting specimen is usually the most concentrated specimen and thus is preferred for
microscopic examinations and for the detection of abnormal amounts of constituents.
Koruyucu kullanılmaz veya sogukta saklanmazsa urine samples can go bad: Glukozun bakterilere yıkımı, ürenin
amonyağa dönüşümü, ph artışı, fosfatlarınn birikimi, ürobilinojenin ürobiline dönüşümü.

Preanalytic Variations: Pre-analytical variation includes all the steps that occur from test ordering until right before
sample analysis. While the likelihood of variation in any of the three phases is not negligible, the vast majority of
laboratory variation emerges from the many factors affecting laboratory specimens prior to testing.

Pre analytic variations influcing factors:

1. Decision about sample type.

2. Controllable variables: Time of Sampling,Influence of Circadian Rhythm,Menstrual Cycle,Influence of
Diagnostic and Therapeutic Procedures,Diet,Effects of Fluid Intake Before Sampling,Smoking Tobacco,Body
Position and Tourniquet,Muscular Activity,Preparing for Blood Sampling,Stress.
3. Noncontrollable variables: Age, Gender, Race

Influence of Diagnostic and Therapeutic Procedures: Operations,Infusions and transfusions,Punctures, injections,

biopsies, palpations, whole-body massage,Endoscopy,Dialysis,Physical stress (eg, ergometry, exercise, ECG),,unction
tests (eg, oral glucose tolerance test),Immunoscintigraphy,Contrast media, drugs,Mental stress,Ionizing radiation.

Diet: Diet substantially affects the composition of plasma. Has Long-Term Effects and acute effects.
Biological Variation:
Smoking also effects the sample.
Intra-individual biological variation or CVI :The concentrations of most analytes fluctuate around a homeostatic set-
Position: free calcium does not change, whereas total calcium increases by 5% to 10%,58 when changing from the
point within individuals.
supine to the upright position
Between individual biological variation or CVg: Homeostatic point differs between individuals.
Tourniquet: higher pressure obtained in veins leads to the loss of water and low molecular weight substances,
increasing the concentration of proteins, cells, and analytes bound to them. This becomes clinically significant after
1 to 2 minutes. Therefore the tourniquet should be released 1 minute after it has been applied.

General Recommendations: Blood should be drawn preferably in the morning between 7 a.m. and 9 a.m. Fasting
should last for 12 hours, during which only water consumption is permitted. Alcohol should be avoided for 24 hours
before blood sampling. In the morning before blood sampling, patients should refrain from cigarette smoking and
caffeine-containing drinks (tea, coffee, etc.).
Biochemical Analysis of Body Fluids
Extracellular Fluid (ECF): Surrounds all cells in the body.
ECF has two primary constituents: Plasma: the fluid component of the blood. Interstitial fluid: Surrounds all cells
not in the blood.
Body Fluid Types: Cerebrospinal fluid • Serous body fluids ( Pericardial fluid , Pleural fluid ,Peritoneal fluid )• Gastric
fluid • Synovial • Amniotic • Saliva • sweat
Cerebrospinal Fluid (CSF):
 Formation : 70% ventricular choroid plexuses , 30% ependymal lining cells of the ventricles and the
cerebral/subarachnoid space
 Reabsorption:Arachnoid Villi
 Volume: 90 to 1 50 mL in adults , Rate of production 500 mL/day or 20 mL/h.
 Functions of Cerebrospinal Fluid : • Buoyancy and cushioning • Maintenance of a constant ionic
environment • Buffering changes in intracranial pressure • Control of respiration
 Composition of csf : Blood-brain barrier : Selectivity,(Molecular weight, lipid solubility, protein binding.
Water and water-soluble substances such as chloride, CO2 , creatinine, glucose, and urea diffuse rapidly)
 The concentration of substances found in CSF should always be compared with their concentration in the
plasma
Csf collection: lumbar puncture:
 Indications =CNS malignancy , Demyelinating diseases ,Meningeal infection ,Subarachnoid
haemorrhage
 Contraindication = presence of infection at the puncture site.
Lumbar Puncture:
• The most common site : Intervertebral space between lumbar vertebrae L3 and L4
• Typically 10 to 20 mL of CSF is slowly removed into three or four sterile tubes : Normal CSF pressure measured with
a manometer in a patient lying flat in the lateral decubitus position with the legs extended is between 60- and 250-
mm H2O.
CSF Analysis:
1. Pressure Measurement : Normal CSF pressure measured with a manometer in a patient lying flat in the lateral
decubitus position with the legs extended is between 60- and 250-mm H2O.
2. Macroscopic evaluation: • CSF is normally clear and colourless • Blood: Erythrocytes are not normally present in
CSF and may be introduced after trauma to a blood vessel during the procedure (“traumatic tap”) or as a result of
SAH.
3. Cell Count and WBC differential:
 Erythrocyte Count: Newborn : 0-675/mm3 Adult : 0-10 /mm3
 WBC Count: Newborn : 0-30/mm3 Adult : 0-5 /mm3
 Differential Count: Adult : 60% lymphocyte 30% monocyte 2% neutrophil
Newborn: 20% lymphocyte 70% monocyte 4% neutrophil
4. Chemistry : Glucose ,Protein, Other: LDH, albumin, cholesterol etc. •5.Other: Culture, staining, cytology, tumour
markers, electrophoresis, serology.
CSF Protein Concentration:
 As proteins are a large molecule, this molecule does not easily cross the blood brain barrier.
 CSF total protein ranges from: 15 to 45 mg/dL.
 Elevated protein : Meningitis , Haemorrhage ,Multiple sclerosis .
 Reduced protein :Fluid is leaking from CNS.
CSF Glucose Concentration:
 Glucose is present in CSF at a level 60% to 70% of plasma in normal adults.
 The normal range for CSF glucose is 50 to 80 mg/dL, with a normal CSF glucose to serum glucose
ratio of 0.6.
 Elevated Glucose : Hyperglycaemia
 Reduced Glucose: Infections, Impaired glucose transport , Increased CNS glycolytic activity,
Metastatic carcinoma.
Serous Body Fluids:
 The serous body cavities are mesothelial-lined potential spaces that surrounds.
 Fluid between parietal and visceral membranes.
 In lungs: pleura, in heart: pericardium, in abdomen and pelvis: peritoneum.
 This fluid is a plasma filtrate derived from capillaries of the parietal membrane.
 It is produced continuously at a rate dependent on: Capillary hydrostatic pressure, plasma oncotic
pressure, capillary permeability.
 An accumulation of serous body fluids : Serous Effusion
 Serous effusion can be transudate and exudate.
Transudate: 1. İncreased hydrostatic pressure. 2. Decreased plasma oncotic pressure. Transudate is bilateral.
Generally in congestive heart failure, hepatic cirrhosis and hypoproteinaemia.
Exudates: 1. İncreased capilary permeability. 2. Decreased lymphatic resorption. Generally in infections like bacterial
pneumonia and tuberculosis, Neoplasms, Rheumatoid Disease.
Microscopic Examination:
 Cell counts: Total cell counts may be performed with manual or automated devices
 Leukocyte counts have limited utility in separating: transudates and exudates.
Differential Leukocyte Count: Examination is commonly performed on a stained smear. Transudates: Mononuclear
cells, neutrofils below %25. Exudates: Neutrofils higher than %25.
Synovial Fluid: Synovium refers to the tissue lining synovial tendon sheaths, bursae, and diarthrodial joints, except
for the articular surface. Synovial fluid (SF) is an ultrafiltration of blood plasma combined with hyaluronic acid
produced by the synovial cells. Examination of the SF is essential to distinguish infectious from non-infectious
arthritis. (specially in crystal-induced and infectious arthritis.)
Synovil fluid tests: • GROSS EXAMINATION • MICROSCOPIC EXAMINATION ( Total Cell Count , Differential Leukocyte
Count , Crystal Examination, Gout: Monosodium Urate Crystals) • CHEMICAL ANALYSIS (Glucose, protein, enzymes)
Monosodium Urate Crystals in Synovial fluid.
Saliva:
 Saliva is produced as an ultrafiltration of the plasma by the salivary (parotid, submandibular, and
sublingual) glands .
 Healthy adults produce 500 to 1500 mL saliva per day.
 Ultrafiltration of blood plasma : Contains proteins, hormones (Enzymes (e.g. alpha amylase,)
,Steroid hormones ,Cardiac markers )
 Interest in saliva as a diagnostic medium has progressed because it offers some advantages over
serum
 Advantages : Collected non-invasively with minimal training .enables an approach for the screening
of large populations
 Disadvantages - challenges : including circadian variations of hormones present in saliva. Analytes
are 1000-fold less than those in blood .Stimuli such as smell and taste affect saliva production and
secretion. Salivary composition is also influenced by the collection method.
Laboratory Testing for Clinical Hematology
Hematology is the study of blood cells and coagulation.
Erythrocyte parameters: Hemoglobin, hematocrit, erythrocyte count, erythrocyte indices
Erythrocyte indices: Mean Cell Volume (MCV) ▪ Mean Cell Hemoglobin (MCH) ▪ Mean Cell Hemoglobin
Concentration (MCHC)
Leukocyte parameters: 1. Leukocyte Counts • 2.Differential (Both % and absolute number) : Neutrophil (Polymorph
nuclear Neutrophilic Leukocyte; Segmented Neutrophilic Granulocyte) ▪ Lymphocyte ▪ Monocyte ▪ Eosinophil
(Eosinophilic Granulocyte) ▪ Basophil (Basophilic Granulocyte)
Platelet parameters: 1 Platelet counts •2. Platelet indices: Mean platelet volume (MPV) ▪ Platelet packed cell
volume (PCT) ▪ Platelet Distribution Width (PDW)

Diagnostic tests: Hematology principles: Hemoglobin, hemotocrit, cell count (RBC,WBC,Platelet), Cell indices (RBC (MCV, MCH,
MCHC , RDW), Platelet(PDW))
 Periodontal disease
 Screening tests ( Cushing (Cortisol testing) , Sex steroids, Neoplasm :( • Breast Cancer • Prostate Hemoglobin: Hemoglobin (Hb), the main component of the red blood cell (RBC), is a conjugated protein that serves
Cancer)) as the vehicle for the transportation of oxygen (O2 ) and carbon dioxide (CO2 ). • When fully saturated, each gram of
 Drug analysis Hb holds 1.34 mL of O2 . • The normal hemoglobin value in ▪ Males is 13.5–17.5 g/dL; ▪ Females 12.0–16.0 g/dL. It is
measured by spectrophotometric method.

Salivary collecting tools: Drool collected in a sterile specimen container , Salimetrics oral swab ,Salivette cotton and Hemotocrit(hct): Hct is the portion of blood occupied by RBCs (%). • The normal hematocrit value is 37–47% . Can be
synthetic device , Greiner Bio-One saliva collection system , OriGene DNA collection device , DNASal collection measured by: Directly with centrifugation ▪ Calculated from the RBC count and MCV as Hct = (RBC × MCV)/1 (Optic
device. Method) ▪ Measured directly by some instruments (Impedance Method)
Gingival Crevicular Fluid: Gingival crevicular fluid (=sulcular fluid): Inflammatory exudate derived from the
Cell count methods: Manual , Automated, Electrical Impedance, Light Scattering , Radiofrequency Conductivity
periodontal tissues.
Electrical impedance: Cells passing through an aperture through which a current is flowing cause changes in
Composition:
electrical resistance ▪ This is counted as voltage pulses. ▪ The pulses, which are proportional in height to the volume
 Individual proteins , of the cells, are counted. ▪ This is the Coulter principle.
 Specific antibodies antigens ,
 Enzymes. Light scattering: In the electro-optical analysers a light sensitive detector measures light scattering. • A focused light
 Cellular elements (Epithelial cells, leukocytes, bacteria), beam illuminates a small area of a flow cell. • Cells are rapidly injected in single file through the illuminated area
 Electrolytes (Na:k ratio in gcf is 3:9 (Plasma 28:1), Fluroide, calcium , iodine and phosphorus) where the cells intersecting the light beam scatter light in all directions in a manner that is measurable and unique to
 Organic compounds : Carbohydrates: e.g. Glucose hexosamine and hexuronic acid , Proteins: The each cell type.
total protein content of gingival fluid is much less than that of serum ,Immunoglobulins ,Cytokines
In light scattering: • Forward, or 0 degree scatter : Correlates with the size of the cell. • A 90-degree side scatter
(lobularity): Corresponds to the granularity of the cytoplasm.
Methods of Collection:
Erythrocyte indices: (RBC)
• Absorbing paper strips
MCV, Mean corpuscular volume: Average volume (size) of the RBCs • Can be ▪ Directly measured from the RBC
• Pre-weighed twisted threads
histogram ▪ Calculated as MCV (fL) = 10 × Hct (%)/RBC.
• Micropipettes
Anemia can be classified based on MCV ▪ Microcytic anemia if its below 80, macrocytic if its higher than 100, in
• Crevicular washings between its normal.

MHC, Mean corpuscular hemoglobin: İs calculated as hb x 10/RBC.

MCHC , mean corpuscular hemoglobi concentration : is the average Hb concentration per unit volume of RBCs,
expressed as a percentage. İf its lower than 31 g/dl its hypochromic, if its higher than 36 g/L you should check for
spherocytes or errors in measurement, if its inbetween its normochromic.
A decrease in MCHC is typically used as an indicator for iron deficiency. • Increased MCHC is a clue to increased
spherocytes, as seen in either hereditary spherocytosis or autoimmune hemolytic anemia
RDW, Red cell distrubition width: Direct measurement of RBC size variation • Reference range: 11.5-14.5% • It
reflects the degree of anisocytosis. ▪ Increases in nutritional anemias • For example, a larger RDW is present in iron
deficiency anemia, folic acid deficiency, ▪ Normal in thalassemia traits, chronic disease, aplastic anemia.
Reticulocyte parameters: Reticulocyte count , Immature reticulocyte fraction , Reticulocyte Hb content.
Reticulocyte: Reticulocyte includes RNA , Normal reticulocyte count %1-2 of RBC count. The reticulocyte count
reflects the state of RBC production. An increased reticulocyte count is indicative of an RBC peripheral
consumptive/destructive process such as hemolytic anemia . A decreased reticulocyte count is a clue to a production
defect.
Platelets: Platelets are cytoplasmic fragmentations from a megakaryocyte. Key cellular elements in the hemostatic
process. Light blue to colourless .Volume: 7.8 – 11 fL.
EDTA-Dependent Pseudothrombocytopenia
Platelet Satellitism Pseudothrombocytopenia
Platelet indices: Mean platelet volume (MPV) ,Platelet packed cell volume (PCT) , Platelet Distribution Width (PDW).
Peripheral blood smear: Wright-Giemsa stain is commonly used • Inspection of cell morphology • Magnification
with ▪ x10 ▪ x40 ▪ x100.
Erythrocytic disorders: Anemia, Polycytaemia.
Iron Deficiency Anemia (IDA):
Causes :Chronic Blood Loss (heavy menstruation, intermittent GI bleeding, etc.) ▪ Increased Need (periods of rapid
growth, pregnancy) ▪ Inadequate intake (diet) ▪ Impaired absorption (malabsorption)
CBC in IDA: ▪ RBC Count: Decreased ▪ PLT: Variable (increased in chronic bleeding) ▪ Hb: Decreased ▪ Hct: Decreased ▪
MCV, MCH, MCHC: Decreased ▪ RDW: Increased.
Iron studies in IDA: ▪ Serum Iron: Decreased ▪ Ferritin: Decreased ▪ Transferrin: Increased ▪ Transferrin Saturation:
Decreased ▪ TIBC: Increased.
Beta-Thalassemia: β-thalassemia is usually due to point mutations in the β globin genes. • These point mutations
cause production of β globin chains to be reduced (β+) or abolished completely (β0).
CBC of b thalassemia: Hb: Decreased ▪ RBC: Normal to Increased ▪ MCV/MCH/MCHC: Decreased ▪ RDW: Normal ▪
RETIC: Normal to Increased • Hypochromic, microcytic RBCs.
Sickle cell disease: Homozygous Hemoglobin S disease • A genetic mutation in the β globin chain results in the
production of abnormal hemoglobin S. • Red blood cells have normal morphology under normal conditions • Under
hypoxic conditions hemoglobin S polymerizes and causes the red blood cell to assume the characteristic sickle shape.
▪ Low oxygen saturation ▪ Decreased pH ▪ Increased 2,3-BPG ▪ Dehydration.
Megaloblastic anemia :Occurs when there are defects in DNA synthesis that cause problems with blood cell
production and maturation • All cells are affected, not just red blood cells • Vitamin B12 (cobalamin) and folate (folic
acid) are common causes • Myelodysplastic syndrome • Hypothyroidism
Vitamin B12 Deficiency : Megaloblastic anemia
CBC of vit 12 def. ▪ RBC, WBC, PLT, Hb, Hct: Decreased ▪ MCV: Usually > 110 fL ▪ MCH: Increased ▪ MCHC: Normal ▪
RETIC: Normal to decreased
Erythrocyte Sedimentation Rate: Erythrocyte sedimentation rate (ESR) is a useful but nonspecific marker of
underlying inflammation. • When well-mixed venous blood is placed in a vertical tube, erythrocytes will tend to fall
toward the bottom. • The length of fall of the top of the column of erythrocytes over a given interval of time is called
the ESR.
Several factors are involved in sedimentation rate: 1. Plasma Factors: Elevated levels of fibrinogen. 2.Red Cell
Factors: Anemia increases the ESR because the change in the erythrocyte/plasma ratio favours roule aux formation.
Indications of sedimentation rate: Hematologic malignancy ▪ Systemic diseases ▪ Autoimmune diseases •
Sample Collection of sedimentation rate: ▪ Long – Black Cap Na-Citrate tube ▪ Blood / anticoagulant ratio is
important: ¼
Sedimentation rate should be Analysed immediately.
Pre-analytic Phase for cbc : • Draw blood collection tubes in the correct order to avoid cross-contamination of
additives between tubes. • Good laboratory practice includes that a correctly filled test tube is one of the
requirements for precise analysis. • Enough mixing is important.
Laboratory tests for assesing of blood diseases:
Hemostasis : The mechanism of preventing excessive blood loose after a traumatic injury. • The mechanism leads to
the production of a hemostatic plug at the site of an injury
Primary Hemostasis • The term primary hemostasis refers to platelet reactivity at the site of vessel injury • Primary
hemostasis is a pro-coagulation clot forming process associated with the initiation and formation of the platelet
plug.
When injury occurs : Blood vessels at the injured site constrict and attract the circulating platelets , The platelets
aggregate in large number and link with one another to form the platelet plug. • Von Willebrand’s Factor (vWF)
enhances the sticking.
Primary hemostasis defect is associated with bleeding at the time of surgery and oozing that continues beyond 24
hours postoperatively.
Secondary Hemostasis: Clot forming process , Plasma coagulation factors plays role.
Tertiary hemostasis: Clot Resolution . Terminate activity of a number of enzymes of the coagulation ,Activation of
fibrinolytic systems
Normal hemostasis is divided into three parts (1) Platelets (2) Plasma proteins (3) Blood vessels
Platelets: Anucleate fragments of the megakaryocyte • Platelets circulate in peripheral blood for approximately 7 to Bleeding Time: Ivy Method: The patient's arm is positioned at the level of the heart • Blood pressure cuff inflated to
10 days with approximately • Have a important role for «Primary Hemostasis». 40 mmHg • standardized device is utilized to make a 10mm long and 1mm deep incision on the volar forearm •
Using a timer the blood is blotted twice a minute. The time stops when there is no further bleeding after blotting.
Evaluation of platelets: Platelet Count (Manual or automated platelet count, Peripheral Blood Smear , Count ,
Morphology ) • Platelet Function Bleeding Time Potential Diagnosis :• Thrombocytopenia (below 50.000/mm3) • Von Willebrand Disease (vWD) •
Disseminated Intravascular Coagulation (DIC) • Severe reduced fibrinogen • Hereditary Platelet Disorders
Platelet Related Disorders: • Thrombocytopenia/platelet deficiency • Platelet dysfunction • Thrombocytosis
Bleeding Time Interpretation :• It has proven a poor predictor of clinical bleeding • Not recommended for surgical
Thrombocytopenia:
evaluation • With the adoption of modern platelet function analysers, this test has largely been abandoned.
The normal platelet count 150 -400 ×109 /L
Coagulation Related Tests • PT : INR • aPTT • Fibrinogen • D-Dimer • Thrombin Time • Factor analysis • Protein C
< 150 ×109/L Thrombocytopenia and S

> 80 ×109 /L minimal bleeding risk Required sample for test: PT • aPTT • Fibrinogen • Factor analysis • Protein C and S. USED SODİUM CİTRATE.

< 20 ×109 /L severe bleeding risk (Spontaneous bleeding). For genetic analysis: factor v leiden, prothrombin gene mutation. USED K2 EDTA.

Causes: Drugs such as heparin, chemotherapeutic agents, and alcohol • Leukaemia, lymphoma, or bone marrow Sample Preparation : Sodium citrate plasma: Centrifugation 1500’g 10 min
tumours • HIV, mumps, rubella, or parvovirus infections • Acute or chronic liver diseases the leading cause for an
Prothrombin Time (PT): • Extrinsic + Common Pathway • Tissue thromboplastin + Factor VII + Ca+2 : Factor X •
enlarged spleen • Idiopathic Thrombocytopenia Purpura (ITP).
Factor V • Factor II » Fibrinogen • Fibrin
Idiopathic Thrombocytopenia Purpura (ITP): Autoimmune destruction of the platelets by IgG antibodies . ITP is most
International Normalized Ratio (INR) :• INR is a simple ratio.  There is no unit of INR
common in children and young adults.
Warfarin :• Oral Anti-coagulant  Vitamin K antagonist  Blocks γ-carboxylation of glutamic acid residues  Factors
Thrombocytopenia Symptoms and Signs: • Easy bruising and easy bleeding • Small, superficial bruises; petechiae
II, IX, X, Protein C and S  Decreased functionality of the coagulation factors  Prolonged PT  Follow up with INR
and bleeding mucus membranes are common
Warfarin: 1. Deep vein thrombosis 2. Atrial fibrillation. 3. Pulmonary embolism. 4. Replacement of the artificial
Evaluation of Platelet Count : For surgery 50 x 109 /L prior to major surgery , 100 x 109 /L prior to surgery involving
valves. 5. Some cases of heart failure.
the brain or eyes
Prothrombin Time (PT) Indications :1. PT and PTT are advised to find the cause of unexplained bleeding or blood clot
In the presence of significant thrombocytopenia, the CBC with platelet count should be assessed prior to dentistry
formation. 2. INR is advised to monitor the thinning drug’s medication like warfarin therapy. 3. Routine health
The test obtained should have been done within the past seven days and usually confirmed on the day of surgery in
screenings. 4. To assess to measure the success or failure of a medication or treatment plan.
very severe cases following replacement therapy
Partial Thromboplastin Time (PTT) :• Formal name:  Activated Partial Thromboplastin Time (aPTT) • Intrinsic +
For outpatient oral surgery or periodontal surgery : Platelet count must be above 75 x 109/L , For major dental
Common Pathway.
surgical procedures should be must above 100x 109/L
Partial Thromboplastin Time (PTT) :1. Mixture of a negatively charged surface + Phospholipid + Anticoagulated
Platelet functions: Adhesion, aggregation, secretion, support of plasma coagulation, clot retraction, support of
patient + 2. Calcium.
damaged endothelium.
Heparin: • Heparin binds to antithrombin III and increases its ability to inhibit thrombin, factor Xa • The
PLATELET FUNCTION DISORDERS • Platelet function is assessed by the capacity of the platelets to adhere/stick to
anticoagulant effect of heparin is commonly monitored by prolongation of the partial thromboplastin time (PTT).
one another. Platelet count is normal but platelets are dysfunctional • Patients may be asymptomatic; however, the
majority who are diagnosed present with easy bruising and mucocutaneous bleeding Indications for heparin: 1. Used to monitor the heparin therapy and control its dose. 2. It is part of the coagulation
panel workup 1. Before the surgery. 2. Evaluate abnormal bleeding
Drugs Associated with Platelet Dysfunction • Drugs ( NSAIDS, aspirin, Dipyridamole, clopidogrel (Plavix) Ticlopidine
(Ticlid) )• Exception of the NSAIDS, irreversibly and permanently affect the entire life span of the platelets ,which is
about 7–10 days • NSAIDS is temporary and lasts until the drug clears the system, which is about 24 hours.

Minor procedures like amalgams, composites, prophys, can be done without stopping any one of these drugs •
Some procedures needs stopping these drugs . Always consult with the patient’s MD before you plan to stop any of
these drugs

Von Willebrand’s Disease (vWD) • Deficiency of the von Willebrand’s Factor (vWF) • vWF promotes : the sticking of
the platelets to the injured vascular endothelium AND Sticking to platelets to form a mesh on which fibrin gets
deposited

Bleeding Time: • Test for the adequacy of primary hemostasis. Screening test for vascular (end) diseases and
platelet function • Abnormalities would require further evaluation with a focus on the coagulation pathway of
interest. A standardized cut was made on the volar surface of the forearm • The resulting drops of blood were
blotted with filter paper • The duration it took for the bleeding to stop constituted the bleeding time.

Factor Assays: • The most commonly performed factor assay is the one-stage factor assay • The assay is performed
on dilutions of patient plasma that are then mixed 1 : 1 with factor-deficient plasma.  Factor-deficient plasma is
deficient in a single factor but contains essentially 100 IU/dL of all other factors..
Haemophilia :• Inherited in an X-linked recessive • Haemophilia A factor VIII (8) • Haemophilia B factor IX (9).
Lupus Anticoagulant :• Immunoglobulin that binds • Phospholipids • Proteins associated with the cell membrane •
Cause an thrombosis (increase in inappropriate blood clotting)
Mixing Test :• Mixing studies are useful when clotting time (eg, aPTT, PT) is unexpectedly prolonged outside the
reference interval. • Mixing studies are performed on abnormally prolonged clotbased assays such as aPTT and PT. •
The purpose of mixing studies is to determine whether prolonged clotting times are due to factor deficiency or
inhibitor activity.
Fibrinogen: • Coagulation ultimately depends on the conversion of fibrinogen (Factor I) to fibrin monomer by
thrombin. • The Clauss method  Excess thrombin is added to the diluted plasma. • N 180-350 mg/L.
Increased level of fibrinogen is associated with: 1.increased risk of coronary heart disease. 2.Stroke. 3.Myocardial
infarction. 4.Peripheral arterial disease.
Decreased level of the fibrinogen level is seen in: 1.Liver diseases. 2.Malnutrition. 3.DIC (disseminated intravascular
coagulopathy)
ACUTE PHASE PROTEİNS:
Acute-Phase Response • The acute-phase response is the term given to the coordinated series of events that occur
non-specifically in response to infection, inflammation, or trauma • This response can be seen as the host's means of
creating an inhospitable environment for the invading microbe. • Acute-phase proteins are raised or decreased •
Variation must be over 25% in inflammatory conditions.  When there is an increase in an acute phase protein
called positive acute-phase protein.  In the case of a decrease in the acute phase protein, it is called negative phase
protein.
x10 – 100 fold increase : CRP , Serum Amyloid A
x2 – 10 fold increase :,  1-acid glycoprotein , Haptoglobin , Fibrinogen
< 2 fold increase : Ceruloplasmin ,C3
Acute-Phase Response Regulation :• The elevation of acute phase reactants is likely a response to the cytokines:
Interleukin-1, Tumour necrosis factor, Interferon-γ, Interleukin-6. • The total physiologic response includes
induction of fever, recruitment of leukocytes, catabolism of muscle, and a shift in protein synthesis patterns with
reduction in albumin production.
Positive Acute Phase PROTEİNS:• C-reactive protein • Serum amyloid A • Fibrinogen • 1-antitrypsin • 2
macroglobulin • 1-acid glycoprotein • Haptoglobin • Ceruloplasmin • Ferritin • C3, C4 • Fibrinogen
Acute Phase PROTEİNS: • Albumin • Prealbumin (Transthyretin) • Retinol Binding Protein • Transferrin
Acute-Phase Protein (Acute Phase Reactants) • The acute phase proteins (positive) are proteins whose
concentration increases in the plasma, and after the disease episode is over, it decreases and may become normal. •
They are thus presumably of benefit to the organism undergoing infection, inflammation and/or tissue damage..
C-Reactive Protein (CRP) :• CRP is produced in the liver, and its name is derived from its reaction with streptococcal
capsular polysaccharides • CRP is produced as a result of pro-inflammatory cytokine signaling primarily mediated by
neutrophils and monocytes. • It has been proposed that CRP may have value as a diagnostic marker for active
inflammation and infection. • Despite these low concentrations, CRP has major significance as a highly sensitive
acute phase reactant • IL-6, IL-1 and TNF-α trigger hepatocyte mediated synthesis of CRP in response to active
infection or inflammation. CRP concentration is elevated during infection or inflammation as part of the innate
immune response • C-reactive protein is normally present in plasma at a concentration below 5 mg/L. • CRP level
supporting bacterial endocarditis, appendicitis, and active collagen diseases was >10 mg/L.
The function of CRP is felt to be related to its role in the innate immune system. • Similar to immunoglobulin (Ig)G,
•It activates complement • Binds to Fc receptors • Acts as an opsonin for various pathogens
CRP, which is the fastest-rising acute phase reactant and one that returns to normal quickly following successful
therapies • Elevations of CRP can be up to 1000 times normal levels, which greatly assists in detecting abnormal
states compared with the other acute phase reactants.
Serum Amyloid A: • Family of apolipoproteins synthesized in response to cytokines released by activated
monocytes/macrophages • Inducing the synthesis of several cytokines  Increased chemotaxis  Increased
phagocytosis. Synthesized predominantly by the liver • Associates with high-density lipoprotein (HDL) • It is as
sensitive a marker for the acute-phase as C-reactive protein (CRP).  During acute inflammation, serum SAA levels
may rise up to 1000-fold . (SAA) is dependent on the release of the proinflammatory cytokines IL-1, IL-6 and TNF-α
during infection and inflammation.
Erythrocyte Sedimentation Rate (ESR) • Nonspecific inflammation marker • Indirect marker for acute phase
response  Increased Fibrinogen  Increased plasma viscosity • Reported as mm/hr  Vertical replacement of RBCs.
Erythrocyte Sedimentation Rate • Normal range in female is under 29mm/hr and under 22 in male.
ESR can increase with other cases without inflammation:  RBC morphology or size  RBC count  Measurement
related factors
Procalcitonin • The precursor of the hormone calcitonin • Its production is governed by the calcitonin 1 gene (CALC- Albumin Decrease :
1) • Biomarker to aid in diagnosis of bacterial infection or sepsis • Serum PCT concentration in healthy individuals is
Decreased Synthesis • Liver injury • Protein malnutrition
typically <0.1 μg/L
Increased Loss: • Loss from kidneys  nephrotic syndrome • Burn
Procalcitonin • Patients with localised infection have smaller increases of PCT in comparison to those with
generalised sepsis, severe sepsis and septic shock

Systemıc Inflammatory Response Syndrome (SIRS): • Body temperature over 38 or under 36 degrees Celsius. •
Heart rate greater than 90 beats/minute • Respiratory rate greater than 20 breaths/minute or partial pressure of
CO2 less than 32 mmHg • Leucocyte count greater than 12000 or less than 4000 /microliters or over 10% immature
forms or bands.
Inflammation, Infection and related Laboratory Tests
Plasma • Plasma contains about 90 percent water, • 10 percent being made up of  Ions  Proteins  dissolved
gases  Nutrient molecules  Wastes
Differences between Plasma and Serum: plasma has albümin, globülin and fibronogen including other coagulation
factors, while serum has albümin and globülin. TOTAL PROTEİN İS ALBUMİN + GLOBULİN.
Proteins in Human Serum and Plasma • They constitute approximately 4% of the body’s total protein • Functions 
Transport  Regulation of the water balance,  Hemostasis  Defence against pathogens.
Plasma Proteins • Synthesis  Liver • (primary importance in the production of plasma proteins  Vascular
endothelial cells (e.g. vWF)  Lymphocytes (e.g. immunoglobulins) • Some 100 different proteins occur in human
blood plasma
1-antitripsin • α1-band • Synthesized by hepatocytes • Function  Most important protease inhibitor in the
plasma • Inhibition of trypsin and other proteases
Ceruloplasmin • α2-band • Function:  Transport of copper ions • Increases with inflammation • Levels are low in
those with Wilson disease
Haptoglobin • α2-globulin • Synthesized as a single peptide chain by hepatocytes • Binding of Hemoglobin •
Decreases in intravascular haemolysis
Transferrin • 1-band • Plasma transport protein for iron (Fe3+) • Increased in Iron deficiency anemia • Negative
acute phase protein
Complement component 4 (C4) • 1-band • Complement component • Positive acute phase protein • Present in
only fresh samples
b2-Microglobulin • It is the non-covalently bound light chain subunit of class I major histocompatibility complex
molecules present on the surface of all nucleated cells. • BMG is shed into the blood, particularly by B lymphocytes,
and some tumour cells • In addition to renal failure, therefore, high plasma concentrations occur in inflammation
and neoplasms, especially those associated with B lymphocytes.

Plasma Protein Structure • Except albumin all plasma proteins are glycoprotein
Protein Electrophoresis • Proteins (and other electrically charged macromolecules) can be separated using
electrophoresis • Electrophoresis separates proteins accordingly their:  Size  Charges .
Generally, serum rather than plasma is used for electrophoresis of proteins  (To avoid the fibrinogen band at the β-
γ interface.)
Prealbumin Band:  Prealbumin (Transthyretin)  Retinol-binding protein (RBP)
1 Band: • 1-Lipoprotein • 1-Acid glycoprotein • 1-Antitrypsin • 1-Fetoprotein • Transcortin
(CorticosteroidBinding Globulin) •  1-Antichymotrypsin • Thyroxin-binding globülin
Fibrinogen • 2-band  Between  and   Clot formation • Only present in the plasma • Positive acute phase
protein
Complement component 3 (C3) • 2-band • Complement component • Positive acute phase protein • Present in
only fresh samples
Urine and BOS Proteins • Urine proteins  For spot urine samples to evaluate must be corrected with creatinine 
For 24-hr collection volume is important • BOS  Prealbumin, albumin and transferrin are present • Total Protein:
15-40 mg/dL • Albumin10-30 mg/dL  Electrophoresis: • E.g. Multiple Sclerosis

2 Band • Ceruloplasmin • Haptoglobin • 2-Macroglobulin • Lipoprotein • Prothrombin

1-Band • Transferrin • Hemopexin • 1-lipoprotein (apoprotein B) • C4 • Plasminogen • FVII, FV

2-Band • 2 microglobulin • C3 • Fibrinogen

-Band  CRP  Immunoglobulins • IgG • IgA • IgM

Prealbumin (Transthyretin) • Synthesized in the liver, 20 – 40 mg/dL • Function:  Transport of thyroxin and
triiodothyronin T4 and T3 • Short half-life (~2 days) • Prealbumin concentrations are often used as an indicator of
adequacy of protein nutrition (Malnutrition evaluation) • Negative acute phase protein

Retinol-binding protein (RBP) • Carries Vitamin A • Negative acute phase protein

Albumin • Synthesized in the liver 3.5–5.2 g/dL • Half-life of 15 to 19 days • Functions  Oncotic pressure  Amino
acid reservoir  Carries small molecules • fatty acids, bilirubin, bile acids, steroid hormones, pharmaceuticals and
inorganic ions