BASIC LABORATORY

INVESTIGATIONS FOR
FAMILY PHYSCIANS.
DR. J.A. OLANIYI
CONSULTANT HAEMATOLOGIST
Blood Smear - Normal
Blood Smear - Normal
 C.B.C / FBC / Hemogram
• Haemoglobin - 15±2.5, 14 ±2.5 - g/dl
• PCV - 0.47 ±0.07, 0.42 ±0.05 - l/l (%)
– Haematocrit, Total RBC volume - better
• RBC count - 5.5 ±1, 4.8 ± 1 x1012/l
• MCHC - Hb/PCV - 30-36 - g/dl
– Hb synthesis within RBC
• MCH - Hb/RBC - 29.5 ± 2.5 pg/l
– Average Hb in RBC
• MCV - PCV/RBC 85 ± 8 – fl
– RBC Maturation
 RBC disorders (Anemias) :
“Anemia is decreased red cell mass
affecting tissue oxygenation”
* Low Hb <13.5 (males), <11.5 (females)
• Acquired / Congenital disorders:
• Decreased production / Increased loss
 Haemolytic An. Introduction
• Anemia due to Increased RBC destruction
• Decreased life span (<120d)
• Breakdown  ↑Bilirubin (Unconj) 
Jaundice
• Increased RBC production - ↑ reticulocytes
• Low Haptoglobins – Hb carrier proteins.
Polychromasia - Hemolytic An.
 Blood Film Features:

• Abnormal shape
• Polychromasia
• Nucleated RBC
• Plt may be low.
 Clinical Features:

– Pallor mild – mucosal

– Jaundice - Mild fluctuating
– Splenomegaly
– No bile in urine (dark on standing-UBG)
– Pigment gall stones – in chronic forms
– Crisis – aplastic, hemolytic, vascular
– Ankle ulcers
Hemolytic Anemia - Types:
– Immune lysis
• Warm & Cold Ab, Auto & Allo immune
– Mechanical Damage
• Valve, Microangiopathy (DIC), prosthesis, march
– Hereditary Defects
• Membrane, Hb & Enzyme defect
– Infection induced
• Clostridia, malaria, septicemia
 Congenital RBC Disorders:

• Membrane Disorders:
– Spherocytosis, Elliptocytosis
• Hemoglobin Disorders:
– Hemoglobinopathies - Sickle cell, HbC etc.
– Thalassemia Syndromes - , , 
• Enzyme disorders:
– G6PD, PK deficiency
Her. Spherocytosis:
Hereditary Elliptocytosis:
 Laboratory Evaluation:
• Features of RBC breakdown:
– Hyperbilirubinemia
– Increased Urine UBG & Faecal stercobilinogen.
– Low or absent Haptoglobins
• Features of increased RBC Production:
– Reticulocytosis
– Marrow erythroid hyperplasia – bone changes
• Damaged RBC
– Morphology, Osmotic Fragility
– Decreased RBC survival – 51Cr labelling.
– Hemoglobin electrophoresis, enzyme abnormality
 Laboratory Evaluation:
• Intravascular Haemolysis:
– Haemoglobinaemia, Haemoglobinuria
– Haemosiderinuria – Renal tubular cells
– Methhaemalbuminaemia – Schumm’s test.
• Molecular genetics
– Hb Electrophoresis
– Globin synthesis studies.
 Haemolytic Anaemia -Acquired
• Infections:- Mononucleosis, Mycoplasma
• SLE
• Drugs: methyl dopa, penicillin,
cephalosporin, quinine
• Malignancy
• Systemic Diseases like Liver disease,
Uraemia)
Sickle Cell Disease:
 Secondary Laboratory Investigation
Cellulose Acetate Hb Electrophoresis
- A2/C S F A+
Normal
Hb SS
Hb AS
Hb SC
Hb CC
HB AD
Sickle Cells
AIHA
AIHA
Schistocytes: Hemolytic anemia
Hemolytic Anemia - nRBC:
CBC Analyzer Report
 Blood Smear Interpretation:

Normal
Micro/hypo
A B C D
Macro
Target
Sphero
Heinz body
E F G H
Schistocyte
nRBC
Polychrom
Teardrop
I J
 Acquired RBC disorders :
• Decreased Production:
– Aplastic, Hypoplastic anemias
– Deficiency anemias Iron, B12, Folate etc.
– Lack of erythropoiesis - Kidney disease
– Marrow disease, malignancy, radiation
• Increased loss/destruction:
– Blood loss anemias - parasites, bleeding
– Hemolytic anemias - Autoimmune (cold & warm
antibody) mechanical, drugs & toxins.
 Iron Deficiency Anemia:
• Most abundant metal but most common
deficiency..!
• Common in developing world,
• Worm infestation, chronic blood loss, poor
diet.
• Etiology:
• Blood loss – bleeding, parasites
• Poor diet – malnutrition (greens & meat)
• Increased need – Pregnancy, children
 Lowered MCV(micrcytosis)
• Chronic blood loss
• Iron def. Anaemia
• Thalassaemia
• Sideroblastic anaemia(MDS)
• Anaemia of chronic disease.
• NB microcytosis in the absence of anaemia suggests
Thalassaemia trait
• MCV may be normal where folate & B12 def combine
with IDA or in ACD, Acute blood loss, MPD, Bone
marrow infiltration
Microcytic Anemia (IDA)
 IDA
• Low MCV
• Low MCHC
• Raised TIBC
• Low serum Iron
• Low Serum Ferritin
• Bf hypochromasisa, aniso, poikilo, pencil cells
• MCV falls in paralel with Hb. MCV is often lower
than in thal.
• Anemia
Clinical Features:
• Pallor, Weakness, Lethargy
• Breathlessness on exertion
• Palpitations may lead to heart failure - edema

• IDA:
• Angular cheilosis, atrophic glossitis,
• dysphagia, koilonychia, gastric atrophy.
Angular cheilitis & Glossitis
Iron Deficiency Anemia:
 Raised MCV
• Normal is 80-99fl
• Vit. B12 or folate def.
• Myxoedema
• Alcohol or liver diseae
• Occasionally Aplastic anaemia & MDS
• Haemolysis
• Marrow infiltration.
 Megaloblastic anemia:
• Vitamin B12/Folic acid deficiency
• Low DNA synthesis – Nuclear immaturity - less
division – more cell size - Macrocytosis
• Megaloblasts – Abnormal – destruction - RBC
• DNA defect – all cell lines affected -pancytopenia
• Multi System disease – cell division - Epithelia.
• Pernicious anaemia
– autoimmune, VitB12 absorption deficiency - Gastric
atrophy – CNS damage.
 Megaloblastic anemia:

• Vitamin B12/Folic acid deficiency

• Second most common type of anemia.
• Vit B12 Synthesised only by
microorganisms – animal food & dairy Pr
• Folate only in Plant foods – uncooked.

• Vegetarianism ..?
Megaloblastic Anemia :
 Megalobl - Pathogenesis:
• Decreased Vit B12 / Folate
• Decreased DNA Synthesis
• Delayed maturation of erythroblasts (Nucleus)
• Increased cell size (macrocytes)
• Normal Hb content (Normochromia)
• Decreased RBC number(Anemia)
• Decreased WBC number (Leukopaenia)
Megaloblastic Anemia :
CWM-20353-Meg.An
 NCNC Anaemia
• Low Hb
• Normal MCV, MCH, MCHC
• Causes
• Chronic disorders eg Renal failure, RA
• Pregnancy
• Recent blood loss
• Haemolysis
G6PD Def - Heinz bodies:
Heinz Bodies
G6PD Deficiency:
G6PD Deficiency Anemia:
Target cells (Liver Disease/IDA):
 Polycythaemia
• Hb, Hct/PCV, RCM allraised
• PCV>0.52 should always be investigated
• In PRVwbc,plt. Count are raised along with the
changes in RBC
• Pseudopolycythaemia/stress polycythaemia
nWBC, nPlt count, decr. Plasma volume but raised
PCV.
• Up to 15% of patients with PRV will eventually
develop a form of leukaemia which is often resistant
to chemotherapy.
 MCH
• Normal 27-33pg
• Should be interpreted with other RBC
parameters
– Raised MCH
• Folate or B12 def.; Myxoedema
Lowered MCH
• IDA, Thal, Chronic Blood Loss,
 MCHC
• Normal 32-36g/dl
• Lowered McHcIDA, Blood loss.
• Raised MCHC may be seen in the
presence of spherocytes and sickle cells.
 White Cells
• Normal WBC 4-11 x109/L. similar in males
and females, constant through out life in
50% of the population
• Diff WBC should be done with count
>11x109/L
• WBC raerly exceed 50x109/L except in
leukaemias
• Afro-Caribbeans have lower WBC counts.
 Diff WBC
• Normal range x109/L
• Neut 2.5-7.5(60-70%)
• Lymph 1.5-4.0(25-30%)
• Mono 0.2-0.8(5-10%)
• Eos 0.04-0.44(1-4%)
• Basophils up to 0.1(up to 1%)
• Afr-caribbeans have lower Neut counts
 Raised WBC(leucocytosis)
• Commonly found in
• Bacterial infection,pregnancy post
trauma,post haemorrhage,
malignancy,Drugs ( steroids, digoxin,
lithium beta agonist), MPD MI, Renal
failure, gout, DM.
 Lowered WBC(Leukopaenia)
• Viral infections
• Bacterial infections( Overwhelming septicaemia),
brucelosis, typhoid, miliary TB)
• Drugs( thiouracil, miacerin, meprobamate,
phenylbutazone)
• Folate or B12 def.
• Autoimmue Neutropaenia, SLE, Felty’s syn. Post
coronary artery bypass graft(CABG) &
haemolysis
 Agranulocytosis
• Drugs ( antimitotic drugs), antrheumatic
drugs such as gold and carbimazole)
• Some malignancies-leukaemia, NHL, may
present with low wbc.
• Neutropenia (Neutro<1x10*9/L due to
infection(bacterial, viral, TB, Typhoid
Brucelosis); Drugs, Aplastic anaemia, bone
marrow infiltration, RA, SLE
 Eosinophilia ( Eos>6%)
• Allergic reactions( to drugs, parasites etc)
• Polyarteritis, reticulosis, Sarcoidosis, MPD,
leukaemia, Erythema multiformis,
Irradiation, Congenital causes, Dermatitis
Herptitformis
• Lymphocytosis(>45%) in Infectious
mononucleosis, Infectious hepatitis,
Cytomegalovirus, Toxoplasmosis, TB and
Leukaemia)
• Lymphopaenia occurs in HD, TB, post irradiation
• Basophilia as part of generalised leucocytosis can
be a manifeatation of MPD(MF, PRV, CML)
 Monocytosis
• Infectious mononucleosis HD, TB,SBE.
Acute and Chronic Leukaemia,
Lymphomas, Solid tumors, recovery after
agranulocytosis
• Lowered monocyte count found in chronic
infection, glucocorticoid treatment,
infections producing endotoxins.
 Morphological Description of
Neutrophils
• Shift to the Left:- Presence of immature
granulocytes. Occurs in pyogenic bacterial
infection, burns, or haemorrhage
• Shift to the right:- Hypersegmented Neutrophils.
Appearance of Neutrophils with >5 lobes. Seen in
B12, folate Def. Accompanied by macrocytes.
Also in renal failure, or congenital anormaly in the
absence of macrocytosis
• Toxic granulations:- Seen in infections or other
toxic states . It is of no special significance.
• Leukoerythroblastic Anaemia:- Seen in
severe infections,
• MPDs
• Bm- infiltration.
– Bone marrow biopsy is mandatory.
 Pancytopaenia
• All cellular elements are reduced(RBC, Plt,
WBC)
• Usually due to BMF, Premature destruction
of cells. Caused by Malignant disease of the
marrow, Autoimmue Diseases (Ra, SLE),
Increased Splenic activity or destruction(eg
portal hypertension), Aplastic anaemia.
Othrs include PA, MDS, Acute Leukaemia.
 CLL
• Lymphocyte count >15x10*/L
• Fairly common in elderly, insidious in
onset, lymphocytosis is unexplained.
CLL
Acute Leukaemias
ALL L1
ALL L2
ALL L3
AML M0
AML M1
AML M3
AML M4
AML M5
CML
CML
CML BM
MULTIPLE MYELOMA
Myeloma BF
Myeloma BM
LYMPHOMAS
Lymphoid Aggregate BMb
Large cell Lymphoma
NHL
 PLATELETES
• Normal 150-400x10*9/L
• Thrombocytopaenia:- Bm hypolasis or
aplasis, BM infiltration, Vit B12/ folate
def., Immue Thrombocytopaenia, including
drugs(eg thiazides, gold, quinidine,
sulphonamides), Infections, hypersplenism,
Dic, liver disease /alcohol, uraemia, blood
transfusions, Idiopathic.
 Thrombocytosis
• Trauma, infection and
inflammation(chronic inflammatory bowel
disease) ,Malignancies and MPDs, Rebound
thrombocytosis in haemorrhage, haemolysis
& post splenectomy)
• Giant platelets seen any acute illness or
haemorrhage
 NORMAL HAEMOSTATIC BALANCE

Naturally
occurring
Pro-coagulants anticoagulants
and platelets. and good
vascular flow

Thrombosis Bleeding
 Introduction: Road map..
• Haemostasis – capacity to minimize loss of
blood following injury to blood vessel.
• Blood vessel – Coagulation – Platelet act.
• Bleeding disorders – Bv, Plt, Coag.
• Laboratory tests of Haemostasis.
• BT, CT, PT, aPTT, TT, FDP.
• Factor analysis, PLT function, PCR,
Haemostasis overview:
BV Injury

Contact/
Neural Tissue
Factor

Blood Vessel Platelet Coagulation

Constriction Aggregation Cascade
Primary hemostatic plug

Reduced Platelet
Activation Fibrin
Blood flow formation

Stable Hemostatic Plug

 DIORDERS OF
HAEMOSTASIS
APTTk
(KCCT kaolin Cephalin Clotting Time}
>40+-7 requires investigation
Measures intrinsic pathway of coagulation
Prolonged APPT:- clotting factor def like fVIII, fIX,
occasionally XI &XII, vWD, Clottig inhibiting
factors such as present in para-protein & Lupus,
liver disease, After Massive Transfusions
 Prothrombin Time
• Normal range (sec.) is control +-4( Usually
13-15)
• Test Extrinsic PW ie FII, VII,X
• The result is inversely proportional to the
prothrombin content of blood being tested
• Thrombin test
• Normal range (%) 7-17
• May be used to monitor warfarin therapy.
 Plasma Fibrinogen
• Normal range is 1.5-4.0g/L (0.2-0.4%)
• Decreased levels found in liver disease, DIC
• Increased level found in tissue damage or infection,
pregnancy, nephrotic syndrome, in collagen
diseases
• Patients with high plasma fibrinogen level > 3.5g/L
as well as high serum cholesterol>6.2 mmol/L and
a systolic blood pressure >140mmHg have a 12
fold increase of heart attack than those with
fibrinogen level <2.9g/l
 FDPs
• Normal range <10mg/l
• Raised level found in increased fibrinolysis
as in
– Post MI
– DVT
– PE
– DIC
– Liver or Renal failure 2* to DIC
 INR
• INR is PT/Control raised to isi
• INR of 1 represents clotting time for normal
individual
• Relevant in warfarin therapy.
 Thrombophilia
• An inherited or acquired tendency towards
abnormal clotting
• 2-3x more common than bleeding
• Seen in women taking third generation oral
contraceptives and with a positive history of
thrombosis
• Consider thrombophilia inpeople <40 yrs with
recurrent thrombo-embolic disease or Primary
thromboembolic eveont
 Thrombophilia- what to check
• ATIII
• Protein C
• Protein S
• Lupus Anticoagulant & anticardiolipin
antibodies
• Family history+ve
• Other predisposing factors to thrombosis
predispose in 50% of cases
 Inherited Thrombophilia
• Inherited resistance to activated protein C2
 occurs in 7% of the population and
produces a variant of fV(fV Leiden)
when discovered in women indicate a
predisposition to thrombosis in pregnancy.

• Inherited deficiency of ATIII, protein C & S

 Acquired Thrombophilia
• Antiphospholipid Syndrome most
frequent cause. predispose to venous &
arterial thrombosis.
 Who should be investigated for
Thrombophilia?
• Venous thrombo-embolism in <40yrs
• Recurrent venous thrombosis or
thrombophlebitis.
• Venous Thrombosis in unusual site ie
mesenteric or cerebral vein.
• Skin Necrosis eg on patient on warfarin.
• Recurrent fetal loss
• Unexplained neonatal thrombosis.
 Thrombophilia-Investigations
• FBC
• PT
• APTT
• TT
• Reptilase Time
• Fibrinogen concentration
• Mainly to detect polycythaemia, thrombocytosis,
dysfibrinogenaemia, suggest presence of LA
 Haemophilia
• Congenital deficiency -Factor 8 (A) or 9 (B)
• Bleeding – Haematoma, joint etc.
• Gene on X chromosome.
– (Carrier females, Males suffer)
• Prolonged PTT but normal PT.
• FFP or Factor replacement – Life long.
 Routine Investigations:

• Bleeding time – BV, PLT

– ivy template method - 3-8min
• Clotting time – inaccurate – 10-20min
• Prothrombin time –Extrinsic (2,5,10 + 7),
– Acquired diseases, liver dis, warfarin therapy
• aPTT – Intrinsic (2,5,10 + 8,9,12)
– Haemophilia, Congenital.
• Trombin Time: Fibrinogen (common path)
– DIC & Heparin therapy.
• CBC – Plt Count
• FDP – Fibrinogen Degradation Products - DIC
 Warfarin therapy
INTRINSIC PATHWAY EXRINSIC PATHWAY

APTT PT
XII VIIa-Tissuefactor
XI IX
X
VIII V COMMON
Protein C/S II PATHWAY

FIBRINOGEN(I) FIBRIN + (XIII)

These are the vitamin K dependent coag factors.
 INR Calculation
PT ISI
MNPT = INR

eg 22 (1.2)

11 = 2.0 (1.2) = 2.3

28 (1.1)

13 = 2.1 (1.1) = 2.3

 Naturally occurring anticoagulants
INTRINSIC PATHWAY EXTRINSIC PATHWAY

APTT PT
XII VIIa-Tissue factor
XI IX
X AT III
VIII V COMMON

Protein C/S II PATHWAY

FIBRINOGEN(I) FIBRIN + (XIII)

SOLUBLE INSOLUBLE CLOT
 FIBRINOLYSIS
Fibrinogen
Plasmin
Fibrinopeptides, Fragment X

Cross-linked Fibrin Fragment Y

Plasmin
Fragment E
Fragments X,Y,E, & D (D-D)
 FIBRINOLYSIS
• Break down of fibrin clot into small
fragments (FDPs) to be cleared from the
circulation by the RE system.
• Fragment D (also known as D-Dimers) are
Specific for Fibrin degradation.
DIC
 Clinical Manifestations of DIC

Ischemic FindingsORGAN ISCHEMIC HEMOR.

are earliest! Skin Pur. Fulminans Petechiae
Gangrene Echymosis
Acral cyanosis Oozing
CNS Delirium/Coma Intracranial
Infarcts bleeding
Renal Oliguria/Azotemia Hematuria
Cortical Necrosis
Cardiovascular Myocardial
Dysfxn
Pulmonary Dyspnea/Hypoxia Hemorrhagic Bleeding is the most
Infarct lung
Ulcers, Infarcts Massive obvious
GI
Adrenal infarcts hemorrhage. clinical finding
Endocrine
Clinical Manifestations of DIC
 Microscopic findings in DIC

• Fragments
• Schistocytes
• Paucity of platelets
 Laboratory Tests Used in DIC
• D-dimer* • Thrombin time
• Antithrombin III* • Fibrinogen
• F. 1+2* • Prothrombin time
• Fibrinopeptide A* • Activated PTT
• Platelet factor 4* • Protamine test
• Fibrin Degradation Prod • Reptilase time
• Platelet count • Coagulation factor levels
• Protamine test
*Most reliable test
 Laboratory diagnosis
• Thrombocytopenia
– plat count <100,000 or rapidly declining
• Prolonged clotting times (PT, APTT)
• Presence of Fibrin degradation products or
positive D-dimer
• Low levels of coagulation inhibitors
– AT III, protein C
• Low levels of coagulation factors
– Factors V,VIII,X,XIII
• Fibrinogen levels not useful diagnostically
 Differential Diagnosis
• Severe liver failure
• Vitamin K deficiency
• Liver disease
• Thrombotic thrombocytopenic purpura
• Congenital abnormalities of fibrinogen
• HELLP syndrome
 Treatment of DIC
• Stop the triggering process .
– The only proven treatment!
• Supportive therapy
• No specific treatments
– Plasma and platelet substitution therapy
– Anticoagulants
– Physiologic coagulation inhibitors
 ESR
• Measurement varies widely in different
physiological and pathological condition
• Approximate normal using Westergren method
formales=Age/2 and for female =age +10/2
• To obtain good result specimen must be tested
within 4hrs, must remain in vertical position &
must be transported to laboratory immediately
• If refrigerated, allow to warm before testing.
 Raised ESR
• Suggests disease(any acute inflammatory
response)
• Pregnancy
• Anaemia
• Oral contraceptive users
• Very high ESR>100 is found in Autoimmue
diseases, malignancy, serious infection
• False ESR if
• ambient temperature is unusually high,
• tube is not vertical,
• dextran present in blood sample.
 Low ESR
• Heart failure
• PRV
• SCA
• Treatment with steroids
 Plasma Viscosity
• Normal(mPa) 1.25-1.72
• Advantages over ESR include
– Independence of Age, Sex and Hb level
– Plasma can be kept at room temperature for
48Hrs without altering the result.
– Not affected by steroid
– High sensitivity, few false negative results,
– Easily automated and cheap.
 Plasma Viscosity
• Reduced(I.e.<1.5) in situations with low
plasma proteins
• Raised in (>1.8):-In the third trimester of
pregnancy
• Up to 3 in acute or chronic diseases
• >3 strongly suggest Myeloma or
macroglobulinaemia