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Good morning

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Lab investgations in

oral and maxillofacial surgery

Dr.ch.v.rao

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Contents

Hematology:-Estimation of haemoglobin-Total RBC count-Total & Differential WBC count

-Blood group determination-Determination of Haematocrit-RBC indices-ESR

-Absolute eosinophil and platlet count-BT/CT/PT/PTT/APTT-Blood banking techniques

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Clinical microbiology- Various staining techniques

- Culture media

- Diagnostic serology

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Clinical chemistry:

- Organ function tests

- Determination of serum bilirubin

- Thyroid function test

- Tests for diabetes

- Total serum protein estimation

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Routine examination of body

fluids and faeces

- Routine examination of urine

- Routine examination of feces- Examination of sputum

- Examination of cavity fluids

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EFFECTIVE DIAGNOSIS HAS 4 ESSENTIAL

REQUISITES

1.Awareness of pattern of clinical presentation

2.Obtaining a complete history3.Undertaking a systematic clinical examination

4.Formulating an appropriate investigative plan

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Clinical symptoms of some diseases are

non specific, underlying conditions maynot be suspected from the history aloneand at times the physical examination

may be surprisingly unrevealing

Its often the routine investigations thatsuggest the presence of underlying

pathology.

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NATURE OF WORK :

Four major areas in most clinical laboratories.

Hematology

Clinical microbiology

Clinical chemistry

Routine examination of body fluids andfaeces

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Purpose of diagnostic tests:

Help to confirm a diagnosis Monitor an illness

Provide valuable information about the clients

response to treatment

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Diagnostic testing involves three phases:-

Pretest

The major focus of the pretest phase is client

preparation

The nurse must know what equipment

and supplies are needed for the

specific test

What type of sample will be needed

and how it will be collected?

Does the client need to stop oral

intake for a certain number of hours

prior to the test?

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Does the test include administration of

contrast media and if so, it is injected

or swallowed?

Are medications given or withheld?

Is consent form required?

How long is the test?

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Intratest

It focuses on specimen collection and

performing or assisting with certain diagnostic

testing.

The nurses uses of special standard

precautions and sterile techniques

Provide emotional and physical support while

monitoring the client as needed (e.g., V /S,

pulse oximetry, ECG). Correct labeling, storage, and transportation of

the specimen to avoid invalid test results.

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Post test

The focus of this phase is on nursing care of theclient and follow up activities and

observations.

Compare the previous and current test results

Modify nursing interventions as needed

Report the results to appropriate health team

members.

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According to STEINER

The laboratory tests and profiles can be classified into three groups.

Group 1 :Those that play an integral role in confirmingthe diagnosis of tissue changes confronted by the

clinician.

Group II : Those that bear directly on the managementof a patient during surgery

Group III :Those that may have a bearing on overallhealth and management of patient.

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(Group II) tests

1. Complete blood count

2. Urine analysis

3. Blood chemistry

4. Determination of BT/CT/PT/PTT

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Estimation of heamoglobin

Measured commonly to obtain informationabout circulating RBC and the amount

Of oxygen carrying substance they contain

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Sahlis method:Cynomethemoglobin method:

Alkaline haematin method:

Haldens carboxyhaemoglobin method:Oxyhaemoglobin method:

Gasometric determation method

Specific gravity method

Photometric method

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Principle ofSahlis method: When blood is added to 0.1N HCl, Hb is

converted to brown colored acid hematin. The

resulting color after dilution is compared withstandard brown glass reference of Sahli

hemoglobinometer.

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Principle of Cynomethemoglobin method:

When blood is mixed with Drakins reagentcontaining Potassium Cyanide and Ferric

cyanide, Hb reacts with Ferric cyanide to formmethemoglobin which is converted to stableCynomethemoglobin by the cyanide. Theintensity of color is proportional to Hb

concentration and it is compared with a knownCynomethemoglobin standard at 540nm (greenfilter.)

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Normal values Hb, g/dl

Children (upto 1 yr) 11.0 13.0

Children (10 -12 yrs) 11.5 14.5 Men 13 18

Women 12 16

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Clinical Significance

A decrease in Hb is an indication of anemia

Definition : reduction below the normal in the volume

of packed red cells as measured by hematocrit or

decrease in hemoglobin concentration of blood

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Classification

Most common and simple classification which isbased on the morphologic characteristics of cells

is:

Based on size of cells: 1. Normocytic

2. Microcytic

3. Macrocytic

Based on

hemoglobinisation of cells: 1. Normochromic

2. Hypochromic

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Microcytic/hypochromic anemia (decreasedMCV, decreased MCHC)

Iron deficiency (common) Thalassemia (common, except in people of

Germanic, Slavonic, Baltic, Native American, HanChinese, Japanese descent)

Anemia of chronic disease (uncommonlymicrocytic)

Sideroblastic anemia (uncommon; acquired forms

more often macrocytic) Lead poisoning (uncommon)

Hemoglobin E trait or disease (common in Thai,Khmer, Burmese,Malay, Vietnamese, and Bengali

groups

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Macrocytic/normochromic anemia(increased MCV, normal MCHC)

Folate deficiency (common)

B12 deficiency (common)

Myelodysplastic syndromes (not uncommon,especially in older individuals)

Hypothyroidism (rare)

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Normochromic/normocytic anemia (normalMCV, normal MCHC)

Normoregenerative normocytic anemias(appropriate reticulocyte response)

Immunohemolytic anemia

Glucose-6-phosphate dehydrogenase (G6PD)

deficiency (common) Hemoglobin S or C

Hereditary spherocytosis

Microangiopathic hemolytic anemia

Paroxysmal hemoglobinuria

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Hyporegenerative normocytic anemias(inadequate reticulocyte response)

Anemia of chronic disease

Anemia of chronic renal failure

Aplastic anemia*

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Drugs and other substances that have

caused aplastic anemia include the

following:

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Hemoglobin concentration drops in pregnancy due tohemodilution

Increase in Hb concentration occurs inHemoconcentration (reduced plasma volume) due toloss of body fluid in severe diarrhea and vomiting,prolong deprivation of water, excessive use of diuretics

High values are also observed in congenital heartdisease due to decreased O2 supply and also inpolycythemia,.

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Total Erythrocyte Count

Principle:The blood is diluted as 1: 200 with the RBC diluting fluid and

cells are counted under high power objective by using Neubauer

counting chamber.

Sodium chloride 0.5 gm

Sodium sulphate 2.5 gm

Murcuric chloride 0.25 gm

Distilled water 100 ml

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Normal values:

Men 4.5 6 x 106

cells/ cu mmWomen 4 4.5 x 106cells/ cu mm

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Clinical Significance:

At birth, the total RBC count varies from 6.5 7.25million/ cu mm.

There is a steady decline after a few hours and atthe end of 15 days to 1 month there is a slow rise tonormal adult levels.

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Increase in RBC count isobserved in conditions like

Hemoconcentration due toburns,

Cholera, Central cyanotic condition, Emphysema, Pulmonary fibrosis, Cor pulmonale, Living at high altitude, Certain erythropoietin secreting

tumors i.e. Renal cellcarcinoma and hepatocellularcarcinoma

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Decrease in RBC count is

seen in conditions like

Old age, Pregnancy,

Anemic state, Leukemias, Bbone marrow

suppression,

Hemorrhage.

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Total Leukocyte Count

Principle: The blood specimen is diluted as 1:20

in a WBC pipette with the diluting fluid (Turks

fluid) and the cells are counted under low power

by using Neubauer counting chamber.

Turks fluid:

Glacial acetic aciddestroys RBCs

1% Gention violetstains WBCs

Distilled water

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Normal values:

At birth: 10000 25000 cells/ cu mm1-3 yrs: 6000 18000 cells/ cu mm

4-7 yrs: 6000 15000 cells/ cu mm

8-12 yrs: 4500 13500 cells/ cu mmAdults: 4000 11000 cells/ cu mm

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Increase in total WBC count of more than 11000 / cu

mm is called leukocytosis and decrease of lessthan 4000 / cu mm is called leukopenia.

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Causes of leukocytosis:

Pathological: Common for a transient period in infections, certain tumors,

myloproliferative disorders, endotoxemias, hypoxia

Also observed severe hemorrhage and leukaemia

Physiological:

High temperature, muscular exercise

At birth total leukocyte count is about 18000 / cu mm At full

term of pregnancy total count is about 12000

15000 / cu

mm.

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Causes of leukopenia:

Certain viral infections like Hepatitis, HIV.

Bone marrow depression

Anemia

Following treatment with: Glucocorticoids,cytotoxic drugs

Autoimmune disorders and malnutrition

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Principle: The polychromic stainingsolutions like Leishmans contain methyleneblue and eosin. These basic and acidic dyes

induce multiple colors. The cytoplasm of WBCsis stained by acidic dye and the nucleus isstained by the basic dye. The neutral component

of the cells are stained by both the dyes.

Differential Leukocyte Count

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Normal values

Neutrophils : 40 - 75 %Eosinophils : 1 4 %

Basophils : 0 1 %Lymphocytes : 20 45 %Monocytes : 2 - 8 %

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Differential count is useful to identifychanges in the distribution of white cells

and also to determine the severity of a

disease and the degree of response ofthe body.

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Neutrophilic

leukocytosis

Acute bact. Infections, tissue

necrosis caused by MI & burns

Eosinophlia Allergic conditions: asthama, hay

fever, pemphigus v.,malignancies

like hodgkins and non-hodgkins

lymphomas.

Monocytosis TB, bact.endocarditis, malaria,SLE.

Basophilia Rare often its an indicator of

myloproliferative disorder like

CML.

Lymphocytosis TB, Brucellosis, infection caused

by hepatitis A, CMV, EBV,

Thyrotoxicosis.

i i i f i

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Neutropenia Starvation, infections

and toxemias in elderly,

typhoid,malaria,hepatiti

s,hypersplenism,bonemarrow suppression.

Eosinopenia Typhoid, aplasticanemia, pt. on steroids

Lymphopenia Severe bone marrow

supression,immunosupressivetherapy, hodgkins

disease, irradiation.

Monocytopenia Aplastic anemia.

BLOOD GROUPING

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BLOOD GROUPING

Around 30 different common antigens and hundreds of rare ones

21 different systems. Common ones are:

ABO System

Rh system

Lewis system

MNS system

P system

Kell system

Duffie system

Lutheran system

ABO system & Rh system

Karl Landsteiner

1900 & 1940 47

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48

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Demographics

BLOOD TYPE population %

O 40 - 65%

A 35 - 45%

B 5 - 10%

AB 3 - 9%

49

D t rmin ti n f H m t rit

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Determination of Hematocrit

(PCV)

PCV is the amount of packed red blood cells

following centrifugation and expressed aspercentage of the total blood volume.

OR

Percentage of volume occupied by red bloodcells in relation to total volume of blood in

centrifuged capillary tube.

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Principle of Macro Haematocrit method:

when an anticoagulated blood is centrifuged in a

haematocrit tube at high speed, the erythrocytes sediment

at the bottom.

The red cell column is called PCV or haematocrit

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Principle of Micro Hematocrit method:

Blood is centrifuged in a sealed capillary tube and PCV

is determined by a special hematocrit reader.

Used : 1. Pediatric patients

2. difficulty in drawing

Sufficient amount of

blood

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Normal values

Birth 5056 %

1-2 years 3238 %

812 years 3642 %Males 4252 %

Females 3648 %

pregnancy 2337 %

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Decrease in hematocritvalues are observed in

anemias, hydremia

(excessive fluids in theblood) as occurs inpregnancy.

Increase in hematocritvalues are observed in

polycythemia, dehydration,

emphysema, congenital

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Color and opacity of plasma

Yellow - Jaundice

Milky - Lipemia

Cloudy - Multiple myloma

Reddish - Hemolysis

Determination of RBC indices

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Determination of RBC indices

(wintrobes constant)

The main indices calculated are

Mean corpuscular volume (MCV)

Mean corpuscular hemoglobin (MCH)

Mean corpuscular hemoglobinconcentration (MCHC)

Color index (CI)

l l ( )

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Mean corpuscular volume (MCV)

Average volume of a single red blood cell and itis expressed in cubic microns i.e.(femtoliters)

MCV = PCV x 10

Rbcs/Cubic mm

The normal MCV is 90 cum (78 to 90 cum).When MCV is increased, the cell is known asmacrocyte and microcyte means the cell withreduced volume.

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Increased : Macrocytic Anemia

Decreased : Microcytic Anemia

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Mean corpuscular Hemoglobin

(MCH) This is the quantity or amount or content of

hemoglobin present in one red blood cell.

MCH = Hb x 10Rbcs in millions/cu mm.

Expressed in microgram or picogram (pg)

Normol value 30 pg (27-32 pg)

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Increases:

Macrocytic anemia (may also remain normal)

Decreases :

Hypochromic anaemia

M l H l bi

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Mean corpuscular Hemoglobin

concentration (MCHC)

Portion of average red blood cell containinghemoglobin

Amount Of Hemoglobin Expressible In Relation To TheVolume Of One Red Blood cell.

MCHC = Hb x 100

PCV

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Most absolute important value in the diagnosis ofanemia.

Normal value 30% (28-38%)

Decreased :Hypochromic anemia particularly in

microcytic hypochromic anemia

Increased: Hereditary spherocytosis

C l i d

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Color index

It is an expression of the mean Hb content of asingle cell in comparison to that of an arbitrarynormal cell

Color index = Hb%

Rbc%

Normal is 1 (0.85 -1.2%)

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Important in determining the type of anemia

Increased: Pernicious anemia

Megaloblostic anemia

Reduced: Iron deficiency anemia( microcytic &

hypochromic anemia)

Normal: Normocytic normochromic anemia

E th t S di t ti R t

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Erythrocyte Sedimentation Rate

The rate at which the red blood cells sediment tothe bottom of the test tube is known asErythrocyte sedimentation rate.

Buffy layer

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Normal values (Westergrens method)

Male 515 mm after 1 hour

Female 520 mm after 1 hour

Normal values (Wintrobe method)

Male 0

9 mm after 1 hourFemale 020 mm after 1 hour

Normal values (Landau method)

Male 05 mm after 1 hourFemale 08 mm after 1 hour

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E.S.R. determination is useful to determine

the progress of the disease and not specific

for diagnosis of a perticular disease except in

rheumatic fever (minor criteria).


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g

ESR is increased in any condition causing an increasein fibrinogen or globulins like rheumatic fever, multiple

myeloma, kala azar, anemias, high temperature,

malignancies.

ESR is greater in women than in men

During pregnancy ESR gradually increases after 3rd

month and returns to normal in about 3-4 weeks after

deliveryLow blood count tend to accelerate ESR

Drugs : Theophylline, vitamin A, Oral contraceptives

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ESR is decreased in polycythemia, sickle cell anemia,

hypochromic anemia, severe dehydration, gastritis,

c.c.f. and in infants.


D t i ti f Pl t l t

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Determination of Platelet

Count

Principle: The blood is diluted to as 1:200 with thediluting fluid using the RBC pipette. After discardingthe first drop, the specimen is transferred to the

counting chamber. Placing the counting chamberunder the petri dish with a moist filter paper allowsthe platelets to settle and also prevents theevaporation of diluting fluid. When observed

through the microscope, platelets will appear likehighly refractile particles.

Normal Range: 250000 500000 /cu mm

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Normal Range: 250000 500000 /cu mm

Clinical Significance :

Determination of platelets is done in cases of

suspected bleeding disorders.


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g

Thrombocytosis is found in polycythemia vera,following splenectomy and Acute rheumatic fever,Hemolytic anemias

Thrombocytopenia is associated with prolonged

BT and poor clot retraction and also occurs in

aplastic anemia, megaloblastic anemia,

hypersplenism, acute and chronic leukemias, sub

acute bacterial endocarditis etc.

Determination of Reticulocyte

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y

Count

Principle of Supravital staining method: Blood ismixed with the stain which enters the cells and theRNA of the cells is precipitated by staining as dark

blue network or reticulum.

Reticulocyte % = No. of reticulocytes counted X 100

No . of red cells counted

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Normal Range: Adults 0.2 2 %Infants 2 6 %

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Clinical Significance: The number of reticulocytes

in peripheral blood is a reflection of red cellforming activity (erythropoietic activity) of bonemarrow.

Increase in their number indicates hyper activity of

marrow called reticulocytosis seen in acuteblood loss or hemolytic anemia.

Low counts of reticulocytes indicates bone marrowdepression seen in aplastic anemia.

Determination of Absolute

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Eosinophil Count

Principle :Blood is diluted with a special diluting fluid( Hingleman solution) that removes the red cells and

stains the eosinophils red. These cells are then

counted using Neubauer counting chamber.

Normal Range: 40 440 / cu mm

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Increase eosinophil count is associated withallergic reactions, parasitic infections,brucellosis, leukemias etc.

Decrease in eosinophil count suggest hyperadrenalism or Cushings syndrome

Bleeding Time

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Bleeding Time

Normal range: Dukes method: 1 5 minutes

Ivy method: 5 - 11 minutes

Prolonged bleeding time is observed inThrombocytopenia, Platelet function disorders,Vascular abnormalities, Anti-platelet

medication(Aspirin), Von-willebrands disease,Leukemia, Aplastic anemia

Clotting Time

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Clotting Time

Normal range: Lee and White method: 5 - 10 minutes

Capillary method: 1 7 minutes

Clinical significance

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Clotting time is prolonged in case of coagulationdisorders like hemophilia,von willebrandsdisease,afibrinogenemia.

Prothrombin time

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Prothrombin time

(Quick,s method)

The time required for coagulation of citratedplasma after addition of thromboplastin,calcium mixture.

Now it is commonly reported with INR.Ratio of prothrombin time that adjusts for thesensitivity of thromboplastin reagent such thatcoagulation profile is reported as INR of 1.

Normal 10-15 sec

I d

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Increased

Anti Coagalant therapy like heparin

Obstructive jaundice

Cirrhosis of liver

Malignancy of liver

Post partum hypofibrinogenemia Congenital deficiency of I, II, V, VII, & IX

Malabsorption syndrome

Early Vit k def

Warfarin therapy

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If P.T. is prolonged - thrombin clotting time isrecommended.

Prolonged PT & PTT defects in commonpathway, ie both intrinsic and extrinsic

Prolonged PT but not PTT (PartialThromboplastin Time), defect in extrinsicpathway.

Partial thromboplastin time:

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Partial thromboplastin time:

Partial thromboplastin ( Rabbits brain aschloroform extact) is mixed with test plasma andCa++ ions, clot formation takes place.

Normal range: 15- 35 secs.

Cli i l i ifi

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PTT is a sensitive measure for the factorsrequired in intrinsic pathway. This is

increased in deficiency of factor I, II, V, VIII, IX,X, XI, XII also in SLE.

A ti t d ti l th b l ti ti

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Activated partial thromboplastin time

Principle:partial thromboplastin ( brain extract in

chloroform ) is incubated with kaolin

(factor XII, contact factor activator.) the clottingtime of plasma is determined after

the addition of Ca++ ions

Normal range: 35-40 sec

Cli i l Si ifi

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Prolonged in1. haemophilia

2. vit. K deficiency

3. liver diseases

4. presence circulating anticoagulant

5. D.I.C.

Shortened in

1. malignancies except those of liver

2. immidiately after acute hemorrhage

PLASMA THROMBIN TIME

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PLASMA THROMBIN TIME

This is the time taken for plasma to clot whencommercially available thrombin is added.

Measure of fibrinogen level. Also affected by excess

of heparin and other anticoagulants.

Normal range: 15 20 secs

Cli i l i ifi

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Prolonged

Afibrinogenemia

Chronic liver diseases

Heparin administration

Multiple myeloma.

E d f t I

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End of part I

MILES TO GO

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