BLOOD GROOPS, BLOOD TRANSFUSION AND BLOOD

INVESTIGATIONS:

When blood transfusion from one person to another were first attempted
the transfusions were successful only is some instances.

After immediate or delayed agglutination and hemolysis of RBC

occurred, leading to typical transfusion reactions leading to deaths. Soon it was
discovered that the blood of different people has different antigen and immune
properties, so that antibodies in the plasma of one blood react with antigens on
surfaces of red cells of another blood.

This is referred to as mismatching of blood.

Antigens-Antibodies and blood groups
In the RBC membrane several classes of antigen are present, for clinical
purposes 2 classes are most important

1) ABO System
2) Rh system

Both systems were discovered by karl Landsteriner in 1900 and 1940

respectively.

ABO SYSTEM:
- The cell membrane of RBC contain antigen called agglutinogens.
- There are 2 such agglutinogens A & B, so the human beings
according to ABO system can be divided into 4 groups.
- Persons whose RBC membrane contains A agglutinogens such
persons are of the category of blood groups A
- similarly persons with. B agglutinogen in their RBC membrane and
are of category of blood group B
 - people with. both A & B in their RBC membrane and are of AB
blood.
- People have no agglutinogens in their RBC membrane and are of O
blood group

Agglutinins of ABO system.

- Plasma contains, agglutinins with are antibodies, (belonging to class
of alloantibody or isoantibody)
- Two classes of antibodies (anti A )
( anti B)
- Anti A agglutinins attacks he A agglutinogen.
i.e. when group A blood is admixed with fluid containing and
agglutinins RBC containing A agglutinogen, are agglutinated.

The Clumped RBCs can

1) Block an arteriole producing blockade of blood supply.
2) Undergo hemolysis producing jaundice or free Hb blocking renal
tubules.

The human plasma can be of 4 types

- those containing alpha,beta and both alpha and beta
.
Blood gps.
Name of blood RBC Membrane Plasma
Group contain agglutinogens containing
Agglutinin.

A A Anti B

B B Anti A

AB A&B Nil

O Nil Anti-A
Anti-B
 Matching-mismatching during transfusion, in ABO system.

In a mismatched blood transfusion donors RBC are lysed but the

recipients RBC’s remain unharmed.

In ABO System group o is called universal donor and AB gp is called

universal recipient provided no Rh incompatibility occurs.

Rh system
Rh owes its name to rhesus monkey in whom Landsteiner and Weiner
discovered this factor.
Only factor D is impt. clinically ie when Rh D is present clinically
person is said to be Rh +ve . When Rh D is obsent it is said to be Rh-ve (10%)

Other systems:
Apart from clinically most impt systems 100 well recognized, systems
are in use eg. Dufty, kidd, kell, Lewis, and MNS system.

Cold & warm antibodies.

alpha & beta agglutinins, Rh antibodies can attack their corresponding
antigen at body temp. and hence they are called warm antibodies.

Antigen antibodies r/n also takes place at 5-20 C hence they are called
cold antibodies.

In very old persons living in cold environments can develop idiopathic

cold agglutinin disease where RBC’s can clump spontaneously is the finger ,
locally in the fingers can drop to very low values.

Nature of antigen and antibodies.

ABO system.

A or B antigen and are carbohydrates attached to a substance H. ( hence

also called ABH system) H substances is composed chiefly of glycoprotein or
glycophingolipid.
Rh antibiodies belong to IgG class of antibodies, and therefore can cross
placenta.
A & B antigens appear in various body fluids such as gastric and
salivary juice. Blood products for transfusion therapy.

A) The usual donation is 450 ml of whole blood i. may be

maintained as such or separated into components.
B) In special circumstances hemapheresis may be used to collect
particular components (eg. Platelets, plasma, granulocytes). This
procedure involves removal of whole blood by phelobotomy with
separation of desired components and return of other components
to the donor.
C) Anticoagulant preservative solutions.

These are substances that are added to blood collection system to

prevent clotting and provide nutrients for continued metabolism of
cellular products during storage.

Commonly used CPD (Citrate phosphate, dextrose) and CPDA1

(with. adenine as subtract for ATP)

Citrate serves are anticoagulant by body Calcium

Storage condition and shelf life.

A)Whole blood and RBCs – 1-. 6 C for 21-42 days depending a preservative
used.
B) Platelets – 20 – 40C for 5 days.
C) FFP and Cryoprecipitate are stored at 18 C for up to 12 months.
D) Granulocytes – 24 hrs. @ room temp. before transfusion.

Individual blood products available for transfusion therapy.

1) Whole Blood:
– One –kit of whole blood contains approx 450 ml of blood + 63 ml
anticoagulant preservative.
– Contains plasma, platelets, leukocytes red cells,
– Used is case of massive blood loss related transfusion depends on blood
loss.
– 1 Unit of whole blood will increase Hg by 1G /dl.
– Not used is patients chronic anemia (PCV is used) /coagulation
disorders.

2) Packed red blood cells.

- Red blood cells prepared by centrifugation have a volume of 250ml

– 350ml. hematocrit of 50% - 80%.
- Uses-Chronic symptomatic anemia who req. increase in red cell
mass but not require vol replacement.
- Patient who are actively bleeding by trauma , surgery.

3) Random-Donar platelet concentrates:

- Each bag contains 5.5 x 10 platelets, in 50 -70 ml plasma.

- Uses Pt who have bone narrow hypoplasia
- Dosage-multiple units of platelets ( 6-8)
- 1 Unit of platelet will increase platelet Count in 70 g adult by 5 x
1000 / microlitre
 - Not indicated in uremia ,Vol willebrand disease.

4) Human leukocyte antigens (HCA) matched platelets.

- HLA matched platelets are collected by hemapheresis from an
individual donor. 1 unit of single donor platelets usually contains 3 x
10” platelets which is equal to 6-8 units for random – donor platelet
concentration
- Post transfusion platelet unit increment
- CI =
(POST TRANSFUSION PLATELELT Count-
PRETRANSFUSIN PLALTELET COUNT.) X bsa
Platetels transfued x 10”

BSA Body surface area.

5) Granulocytes CMC
- Prepared by leukopheresis from a single donor.
- Use profound neutropenia due to myeloid hypoplasia of bone
narrow.
- Septic neonates
- Severe granulocyte dysfunction syndrome granulomatous disease.
- Dose 4-6 days of granulocyte therapy i.e. 1 unit/day for beneficial
effect.

6) Fresh frozen plasma:

- Prepared from whole blood by separating and freezing the plasma I
in 6 hours of phlebotomies.
- Plasma maintained in frozen state contain both labile factors (V ,
VIII) & stable factors ( I, II, VII, IX, X, XI, XII, XIII)
- Single unit FFP ranges from 200 – 250 ml
- Use: Pt with multiple coagulation def/specific coagulative factor
def.
 - Massive transfusion.
- Dose depends in Clinical. Situation
for initial therapy 15 ml / kg (4-6 units) regd.
- not used as – volume expander/nutritional supplement

7) Cryoprecipitate:
- Prepared by thawing one unit of FFP at 4 C
- - Use –Von willebrands discase (Partial defiency of factor VIII.
- Hypofibrinogenemia (acquired as part of DIC syndrome) (if
fibrinogen level is less than 100-120 mg/dl)
- Hemophilia A (factor VIII: C def)
- Factor XIII def.
- Bleeding related to renal disease reason for cessation of bleeding is
not known.
- Dose: Plasma vol.X (desired factor level-in U/ml-initial factor level
in U/ml.
80 U factor VIII. C/ bag of cryoppt.

8) Albumin:
-Prepared from plasma available in concentration of 50 – 250 g/L
- Use – Pt. with. chr albumin depletion due to liver failure.
Hypovolumia in shock.
- Burns-extensive cases
- Dose-desired circulating plasma level 5.0 gl/dl.

9) Immune screen globulin:

–Over 90% of protein is Ig G trace of Ig A & Ig M protein,
- ½ life of i.v. and im have been reported as 18-32 days.
- Use: Primary . Immunodef states such as wiskoh Aldrich syndrome,
congenital agammaglobulinemias
- Dose-0.5-10.0g vials.
10) Rh(d) immune globulins
- Use solution of anti Rh(D) Ig G antibody
- Uses
fetmateranal hemorrhage to prevent immunization in rh (D) ve-women.
-Dose antepartum or postpartum a full 300 mg. dose (in 72 hrs of exposure
to Rh(D) +ve ed cells.

10)Factor VIII concentration

-Used in Patient with hemophilia A
-Dose
-Plama vol X (Desired 10 ml – initial 10 ml) = no of bottles
1U/ Bottle of factor VIII.
12) Factor VI CMC (Prothrombin complex concentrate)
-Uses-Factor IX def (hemophilia B /Christmas disease)
13) Antinhibitor coagulation complex.

14) Other products:

-Antithrombin III
Protein S
Protein C
Fibronectin.

COMPATIBILITY TESTING AND CROSSMATCHING:

Whenever blood is to be transfused appropriate and cross matching test
should be done.
The series of test that must include the foll.
1) Correct identification of donor and recipient
2) A review of pts past history and blood bank rec. for type and presence of
unexpected antibodies
3) ABO & Rh typing of both donor and recipient.
 4) Testing of screen of the donor and recipient for presence of unexpected
antibodies
5) Cross matching of donor red cells with. pts screen.

ABO and Rh. Typing of donor and recipient.

-ABO gp is matched and Rh type is selected with respect to D factor.

-Pts whose cells contain D antigen are given blood with D factor only and
vice versa.

Unexpected antibody screening.

- Testing the pts serum with a panel of gp o reagent red cells that are
known to contain antigens to the most important clinically
significant unexpected antibodies.
- Allows testing of pts serum in advance of actual transfusion,
allowing for selection of rare blood gp.
Cross matching:
1) Saline cross match
2) High protein cross match
3) Antihuman globulin cross match.
4) Enzyme cross match.

Major and minor cross match.

- Major cross match involves pts serum with donars red cells.
- Used to detect unexpected antibodies in pts screen that which. reach
to donors red cell.
- What pt. screen will do to infused blood cell us detrain of infused
cell will lead to transfuses r/n
- In minor cross match, tests donor’s screen with. pts red cells.

Saline Cross match:

 - Mixing screen and 2% - 4% suspension of cells in a saline in a best
tube.
- Incubated @ room temp, centrifuged, observed for presence of
agglutination or hemolysis.
- ABO in compatibility observable.
- Also incompatibity with. P, MNS, Lewis, Wright system observed.

Adverse Effects of Transfusion:

a) Immune-mediated adverse effects.

1) Acute hemolytic transfusion r/ns.

pathogenesis:
• Usually due to transfusion of ABO incompatible
whole blood or RBCs following. a clerical error
eg. Mislabeling of a pts sample for ABO typing)
or transfusion of blood to a pt other than
intended recipient.
• IgM component of naturally occurring anti A or
Anti B antibodies present in the pts serum
agglutinates transfused red cells that have the
corresponding antigens leading to complement
activation and intravascular hemolysis.
• Subsequent involvement of neurooendocrine
and coagulation systems may lead to shock,
acute renal failure and DIC.

Signs and Symptoms:

• Symphonious are variable depending on ant of
blood transfused.
• Fever, chills, chest and backpain,
 • Pain at infusion site
• Nausea, dyspnea
• Hemoglobiurua oliguria
• Bleeding
• Hypotension
• Shock
In an anesthetized pt, he only signs may be uncontrolled hypotension,
hemoglobulinuria and excessive bleeding.

Treatment:

- Transfusion discontinued immediately

- Initial therapy is to control hypotension and promotion of renal blood
flow with. fluids and diuretics
- Prompt evaluation of blood should be done.

PREVENTION:
-Accurate labeling of pt. samples
Accurate identification of recipient at time of transfusion.

2) Delayed hemolytic transfusion r/n

PATHOGENESIS
• Due to pri/sec immunization against red cell alloantigen i.e foreign
antigens nt. Pr on pts RBCS) such as Rh system, Duffy, kidd kell.
• Pri antibody production begins 1-2 weeks foll exposure.
• See response occurs in previously immunized pt.
• Foll re-exposure to Red cell alloantigen a rapid in antibody tile (IgG) is
seen in 5 days.
SIGNS & SYMPTOMS:
- May be subclinial
- In cases of rapid rise in titre of antibody or when antibody activates
complement recipient develops, fever, chills, anemia, jaundice and
hemoglobinemia
TREATMENT:
- monitor renal function in pt. with renal disease.
- Screening test to evaluate immune mediated hemolysis with. direct
antiglobulin test (IgG) antibody screen.

3)Febrile non hemolytic transfusion r/n

Pathogenesis: Occur: Recipient develops agglutinating or cytotoxin antibodies
against antigen on donor granulocytes lymphocytes or platelets.
- More commonly seen in multitransfused pts.

Signs and Symptoms:

• Fever (38 C)
• Chills
• Rigors.

Rx:
• Antipyretics
• Use of in line filters to remove contaminating
leukocytes from RBC’s whole blood/platelets.
4)Non Cardiogenic Pulmonary edema
Pathogenesis:

-rare r/n due to potent leukoagglutinins

Antigen Antibody interaction leads to leukocyte aggregation and trapping in
pulmonary vasculature, causing increased vascular permeability and activation
of complement with generation of anaphylotoxins

Signs and Symptoms:

-Acute pulmonary edema
-Fever and Chills.

Rx and prevention:
-Use of Iv steroids and supportive measures helps in early resolutions of
condition.

5) Allergic transfusion r/n

A) Urticarial r/n
Pathogenesis:
-Seen in ppl with. history of allergy
-Foll transfusion of plasma or cellular blood products
Symptoms are mediated by histamine release IgE or IgG crated mast cells.

Signs and Symptoms.

-Hives & pruritis
No fever.

Rx & Prevention:
-Administration of antihistamine
- Repeated urticarial r/ns unresponsive to medication may warrant removal of
excess plasma from cellular blood products

b) Anaphylactic r/n
PATHOGENESIS:
-Seen in IgA deficient patients( 1 in 700 patients)
r/ns to transfused allergens (penicillin, ethylene oxide, alloantigins) (Eg. C4
albumins)

Signs & Symptions:

Dyspnea
Bronchospasm Occur after only vol
Vomitting is transfused
Diarrhea
Hypotension

Rx:
-hypotension
Admn. epinephrine
Prevention of hypoxia

PREVENTION:
-Pts to enroll in autologous donor programs prior to elective surgery.

6) Alloimmunization:
-Foll transfusion individuals may becomie immunized against a number of diff
alloantigenic components in blood products inlc RBC antigens platelet specific
antigens etc.
a) Alloimmunization to red cell antigens
PATHOGENESIS:
- May occur foll transfusion or in pregnacy
- Found in 0.3- 2% of population
- Pri IgM or sec. IgG immune responses may be detected by rise in
liter of red cell specific antibodies.

Signs Symptons Rx.

-similar to acute or chr hemolytic r/n
PREVENTION:
-Incidence of immunization may be fed by judicious use of red cell transfusion.

Erythroblatosis fetalis (Hemolytic disease of newborn)

-Disease of fetus and neutron child characterized by agglutination and
phagocytosis of the fetus’s rbc.
-In most instances mother is Rh -Ve and father is Rh +ve, the baby has
inherited Rh +Ve antigen from father and another develops ants, Rh agglutinins
from exposure to baby’s Rh antigen.
Incidence:
-Rh –ve another having Ist Rh +ve child usually doesnot develop sufficient anti
Rh agglutinins to cause any harm.
-But 3% of 2nd Rh +ve babies exhibit sign of Erythroblatosis fetalis.
-10% of third babies exhibit the disease
-Incidence increases subsequent prepnancy.

Eff of mother antibodies on the fetus.

-Diffuse through placental membrane into fetus blood.
-Agglutinated RBC’s hemolyze relasing HB into blood.
-Fetus macrophages convert HB into bilirubin
-Baby’s skin becomes yellow (jaundice)
Antibodies can also attack and damage other cells of he body.

Clinical picture of erythroblastosis.

-jaundiced erythroblastic newborn baby is usually anemic @ birth.
-Anti Rh agglutionis circulate in infants blood for 1-2 months after birth
destroying none RBC’s.
-Liver and spleen enlarge due to hyperfunction
-Immature nucleated cells are pushed into circulaltion.
-Due to presence of nucleated blastic RBCs disease is called Erythroblatosis
fetalis.
Anemia is the usual cause of death.
-Children 2nd survive anemic exhibit permanent mental impairment or damage
to motor areas of blain because of ppt of bilirubin in the neronal cells causing
their destrn. This condition is called kernicterus.

Rx:
-400 mol of Rh -ve blood is infused over a period of 1 ½ hr or more hours
whiles neonates own Rh +ve blood is removed.
-Repeated several tinu during Ist wee of life, mainly to keep bilirubin level low
and prevent kernicterus
-By the time Rh –ve cells are replaced infants own Rh +ve cells a process that
requires. 6 weeks, Anti Rh agglutinins that had come from the another will be
destroyed.

Alloimmunization of Platelets:
PATHOGENESIS:
Alloimmunization platelet associated HLA antigens results in refractoriness to
platelet transfusion seen in multiple transfused pts like leukemia, solid tumor or
aplastic tumor
Alloimmunization platelet specific antigens may cause neonatal alloimmune
thrombocytopenia
Alloimmunization to platelet specific antigens may also cause post transfusion
purpose.

Sign and Symptoms:

-Intracranial , gastrointestinal or genitourinal haemorrhage is significant in Ist
24 hrs.
-Pt with post transfusion purpura have a risk of hemorrhage from profound
thrombocytopenia that occurs 5-10 days after transfusion.
Rx & Prevent:
-HLA matched platelets are used
Rx Complication of hemorrhage
&) Graft v/s host disease (GVHD)
Occurs when immunocompetent allogenic lymphocytes are transfused or
transplanted into severely immuno compromised recipient.

Signs and Symptoms:

-Fever
-Liver funct abnormalities
Diarrhea
Erythematous skin rash

Rx & Prevention:
-Steroids
-Antithymocyte globulin
-Methotrexate
-Cyclosporine
-Irradiation of able blood products is currently most eff method for prevention.

b) Infections disease transmission.

Complications due to massive transfusion :

Defined as replacement of at least 1 blood volume with blood
components within 24 hr. period. May be necessary in case of treatment.
Liver transplant or complicated vascular surgery.

1) Haemostatic abnormality
- Manifests coagulapathy & thrombocytopenic purpura
2) Citrate toxicity.
 - Citrate is impt. Component of anticoagulants used for blood
storage. Excessive amount causes cardiac dysfunctions, as a
result of hypocalcaemia.
- CaCl2 or Ca gluconate given to patient, it in blood
3) Hypothermia
- Decrease in body temp – impair citrate metabolism – increase in
affinity of Hb for oxygen-cardiac arrythemias
- Use of blood warmers
4) Acid – base imbalance
- Early finding - metabolic acidosis
- Late finding – metabolic alkalosis (citric lactate – bicarbonate)
- Restoration of BP, and tissue perfusion may rapidly improve
acidosis.

5) Potassium imbalance:
- Hyper kalemia C&P in neonates
-Hyokalemia
Rx – Fresh blood (less than 7 days old) is used

6) Mech. trauma to red cells.

- Causes – faculty blood warmers
- Mech pumps
- Extra corpreal circulation.

D) Other non immunologic complications:

1) Circulatory overload
C/F * Dyspnea
 Orthopnea
 Cyanosis
 Peripheral edema
 Rx * Diuretics
* Oxygen

1) Hemosiderosis
Causes hepatic, renal and endocrinal dysfunction.
Rx - Deferoxamine – Iron-chelating agent.

Hematology Investigations :
1) Hemoglobin
- Hb is determines as gas of Hb / 100ml of blood or gm/dl.
- Reporting Hb as % of (N) value is not satisfactory: there are so
many different methods and each method has its own normal
value.
Sahli 17.3 g/dl
Dare 16.0 g/dl.
Haden 15.6 g/dl.
Wintribe 14.5 g/dl.
Haldane 13.8 g/dl.

Hb (g/dl)
Birth 14.9 :23.7
2 weeks 13.4 - 19.8
2 months 9.4 - 13.0
6 months 10.0 – 13.00
1 year 10.1 – 13.0
2-6 yrs 11.0 – 13.8
6-12 yrs 11.1 – 14.7
12-18 yrs M 12.1 – 15.1
F 12.1 - 16.6

Methods used to determine Hb.

 1. Visual Hb methods – Eg. Dare. Haden, Halden, Sahli method
2. Gasometric methods – Van Slyke apparatus
3. Quantative Spectro photometric methods.
- Oxyhb method
- Heniglobnaaxide method
- Automated hemoglobiometry.

Variations in Ref. Values :

1. Bran B 1988 reported slight decrease in Hb level after age of 50 yrs.
2. When Hb value is decreased normal patient is said to be anemic.
2. Increase in Hb usually results due to increase No. of erythrocytes
(erythrosis) is seen in polycythemia and newborn infants
3. (N) Hb conc. Is higher in higher altitudes than at sea level.

Hematocrit (Packed cell volume).

Hematocrit is a macroscopic observation by which the percentage
volume of the packed RBCs is measured.
Hematocrit is used in evaluating and classifying the various types of
anemia according to red cell indices.

A fast quality control check on Hb results is done by comparing them

with hematocrit results.

Hb x 3 = HCl +/- 3 units.

(N) adult male - 0.42 – 0.50 (42% - 50%)

adult female - 0.26 – 0.45 (36% - 45%)

Red blood cell indices :

These are calculated from the total no. of red cells, the Hb content per
unit volume and the hematocrit.
 The indices are
1) Mean corpuscular volume – defines volume or size of any RBC
2) Mean corpuscular Hb (MCH) – defines wt. Of Hb in any RBC
3) Mean corpuscular Hb conc. Defines Hb conc./color of any RBC
(MCHC)
4) Mean corpuscular diameter (MCD) – quantitative measurement
determines degree of red cell vaiability.

1) MCV = HCl 20 x 10 = ___ H

RBC count x 10IL/L (H=10-15L)

Clinical significance :
- Indicates whether RBCs will appear microcytic,
normocytic or macrocytic.
- MCV < 80 ft – Microcytic
- MCV > 96 ft. – macrocytic
- Most reliable automated index and most eff.
Discriminate for classification of anemia.

2) Mean Corpuscular Hb (MCH)

It is avg. wt. Of Hb content in an RBC in pico gm. (Pg = 10-12g)

MCH = Hb(gldl)x10
RBC count (X1012/l)

(N) – 27 - 33 g.

Clinical significance:

-In case of macrocytic anemias MCH is greater than 50 pg.

- < 20 pg in hypo chromic microcytic anemia.

3) Mean Corpuscular Hb conc.

 - Expression of any Hb conc. Per unit vol. Of packed red
cells
- Expressed in gm / deciliter

MCHC ( g / dl) = MCH x 100

MCV
Or
MCHC (g / dl) = Hb(g / dl)
Hdl (L/L)

(N) = 33 – 36 g (d)

Clinical significance:
- Values < 32 g/dl indicate hypochromia
- As MCHC above 4 0g/ dl – malfunction. Of instrument or error
in
calc because MCHC of 37 g / dl is near the upper limit of Hb
solubility thus limiting the physiologic solubility.

4) Red cell distribution width:

- Ref to degree of red cell variability or anisocytosis
RDW = SD of MCV x 100
Mean MCV

Range of ROW = 11% - 15%

Erythrocyte Sedimentation Rate :

- If blood is prevented from clotting and allowed to settle,
Sedimentation of erythrocyte will occur.
- The rate at which the red cell fall is known, as Erythrocyte
sedimentation rate (ESR)
- This rate depends on three main factors
• The No. and size of erythrocyte articles
• Plasma factors (fibrinogen and globulin)
 • Tech and mech. Factors.

Degree of erythrocyte sedimentation :

1. 1st variable period of gradual falt during the aggregates of erythrocytes
are forming (rouleax formation)
2. Very raid and marked fall of aggregate occurs, constituting the main
ortion of sedimentations of erythrocytes.
3. Erythrocyte aggregates are being packed at the bottom of sedimentation
tube.
Clinical significance of erythrocyte sedimentation rate.
1. ESR is non-specific screening test for inflammatory activity.
2. Infections such as chorea, undulant fever are two exceptions.
3. Increase in carcinoma, leukemia, diseases of bone marrow, degenerative
vascular disease, active rheumatic fever, multiple myeloma, systems
lupus erythromatsis, rheumatoid arthritis, and acute joint.
4. ESR may still be increased after clinical manifestations have
disappeared, showing that the def. Mech. Of body continue to be more
active than (N).
5. Increase in No. of erythrocytes as seen in poly cythemia and failure of
right side of heart tend to cause slowing of sedimentation.
6. Decrease in ESR – decrease in plasma fibrinogen level as in cases of
severe liver diseases.
7. ESR red in viral diseases such as infections mononucleosis and acute
hepatitis probably because fibrinogen production is not increased in
disease in spite of pronounced inflammatory reaction.
8. Other than inflammatory joint disease, ESR is not usually red in color
degenerative joint disease.
- Winrobe & Westergren methods are commonly used to measure
ESR
- (N) ESR – male -0-10mm/ltr
female - 0.20mm / liter.
COUNTING THE FORMED ELEMENTS OF THE BLOOD
(HEMOCYTOMETRY).
Enumeration of the formed elements of the blood is a fundamental
measurement in the hematology laboratory.
The cells counted in routing practice are RBC’s WBC’s and platelets.

Units reported:
Enumerated constituents are to be reported in units per litre of blood.
Hence no. of cells or formed elements actually counted must be converted to
the no. of cells present per litre of blood.

Haemocytometor or electronic cell counter is used to count the no. of

cells.
Clinical significance
1) Leukocyte count.,
• (N) = 4.4 – 11.3 x 109/L
• is called Leukocytosis
• is called leucopoenia.
• Leucopoenia occurs after X-ray therapy, typhoid, malaria, pernicious
anemia, circulation of liver rheumatoid arthritis, hepatitis.
• Leukocytosis may occur in acute infection severe malaria, during
pregnancy, after hemorrhage post operatively, leukemia, appendicitis,
ulcers.
• Child’s leukocyte count greater variation during disease than an adults.
• Leukocyte slightly higher in afternoon
• X after strenuous exercise, emotional strew and anxiety.

Erythrocyte Count :
1. Anaemia is term, with reference to raise in no. of erythrocytes
2. Polycythemia is constitute where erythrocytes are fed.

Clinical significance of platelet Count.

- A count lower than (N) may be associated with generalized
bleeding tendency and prolonged bleeding time.
- A count higher than (N)may be associated with a tendency
towards thrombosis
- Thrombocytopenia ( decrease in platelets) is found in
thrombocytopenic purura, aplastic, pernicious anaemias. patient undergoing
radiotherapy and chemotherapy.
- Thrombocyostsis or raise in platelets can be found in rheumatic
fever, asphyxiation following surgical , spleenectomy, acute blood loss,
leukaemia chemotherapy.

MICROSOPIC EXAMINATION OF PERIPHERAL BLOOD.

1) NEUTROPHILS :
- Most numerous of granulocytes are polymorphonuclear neutrophilic
leukocytes or segmented neclrohilis granulonytes raning from 10-14 mm
diameter.
- Chromatin– irregular arranged is compact mass
- Nucleus is Tabular with elongated nucleus forming No. of lobes.
- Neutrophilia – acute infection, metabolic, chemical, and drug
interaction, acute hemorrhage, malignant neoplasma.

2) Eosinophils
- Granular
- Larger than neutrophils
- Nu occupies small part
- Eosirophilis () allergic, skin disorder, parasictic
infection, brucellotis, hodgkins diseases.
3) Basophils
- same size as Neutrophil
- Nu occupies greater portion of cell.
- Basophilis ( ) – non lymphocytic leukaemai, myeloid
metaplasia and polycythemia vera.
4) Monocytes
- larges leukocyte
- Nu very large, lobulalar, oval notched or polymorphic
- Azur dust seen only in is monocyte.

5) Lymphocyte :

-2types - large (204m)

small (104 m)
- Lymphocytosis ( increases) – acute infections
mononulus and chr infection like TB, brucellosis,
infections hepatitis.

Greater plasma cells.

- occur in certain blood specimens
- not to be derivative of B lymphocyte
- found in measles, chicken pox, scarlet fever,
plasmacytic leukaenia, multiple myeloma

CLINICAL SIGNIFICNCE OF BLOOD CELL ALTERATIONS :

1) alterations in Erythrocyte size
* Anisocytosis - Increased variation in size
* Macrocytosis - large red cells
- 94 m dia
- MCV > r 100ft
- Megaloblastic anaemia.
 * Microcylosis - Small red cells
- < 6.5 um dia
- MCV < 78 ft
- IDA, thalassemia, lead poisoning sideroblastosis
anaemia, iodiopathic pulmonary hemosiderosin.

2) Alterations in Erythrocyte shape

* Discoytes - (N) shape discocyte
* Poikilocytes - greater variation in shape
- ear, oat, teardrop, triangular helmet shaped
fragmented
- Hemolytic states
* Elliptocytes (Oval cells)
- Egg shaped
- Megaloblastic anaemias

* Drepanocytes- sickle cells

- Crescent shaped
- seen in sickle cell anemia
* Codocytes (Target cells).
- showing a peripheral ring of Hb
- as ar of pallor or clearing
- seen in chr. Liver diseases.

* Spherocytes - lesser 6 micrometer ref as microsphercytes

- hemolytic anaemias
Dacryocytes - (teardrop cells)
- tear shaped

3) Alteration in Erythrocyte structure and inclusions.

 * Basophilis stippling :
- prior of dark blue granules distributed evenly through
out red cell.
- Seen in thalassemia prior, megaloblastic anaemia, lead
poisoning

Hollwell – jolly bodies

- round, densely stained purple granules
- Megaboblasltic anaemias

Cabots rings : - thread like red violet strands occurring twisted, ring or
figure of 8 shapes
- seen in megablastic anemia or lead poisoning

4) Morhologic alterations in lenkocytes :

1. Dohle bodies
- round / oval
- small clear light blue stain
- pregnancy, burns, admn. of toxic agents.

2. Aver bodies –
• rods are slender, rod shaped
• needle shaped
• lymphocytic leukemia.
Smudge / basket cells
- Damaged white cells
- CLL

IMMUNO HAEMATOLOGY :
Compatibility testing :
1) Determination of patients (recipients) ABO group
 ABO group of patient is determined by testing the patients red blood
cells and scrum with commercial antisera (anti-A and and anti-B) and cells (A,
B and O) respectively for agglutination.

Antiges and Antibodies in the ABO Group system.

ABO Group Red cell antigers Red cell antibodies
Present Present in serum
A A Anti-B
B B Anti-B
AB A and B Neither
O Neither Anti-A and
Anti-B

Clinical significance :
1) Transfusion of ABO incompatible may cause acute hemolytic
transfusion reaction because of presence of antibody in recipients serum causes
hemolysis of transfused red cells expressing the corresponding antigen.

2) ABO hemolytes disease of the new born may result when

maternal anti-A and anti-B antibodies of IgG isotype cross the placenta and
bind to fetal red cells expressing corresponding antigen.

3) Determination of the patients (recipients) Rh(D) type :

After A and B, the D antigen is the most important red cell antigen is
transfusion practice. This protein antigen is part of Rh blood group system a
complex system that is composed of more than 40 cell antigens.

Other Rh antigens include C, E, c and e.

Clinical significance :
a) Over 80% of Rh D –ve patients who receive a unit of Rh O +ve
RBC will become immunized and produce anti D antibodies.
 b) Anti D antibody is the most common cause of severe hemolyte
disease of newborn. Therefore it is important that only Rh(D) –ve cellular
components immunized Rh(D) –ve females until the end of child bearing years.

4) Detection of clinically significant alloantibodies in the patients

serum (antibody screen; indirect antiglobular test).

Many other blood groups also define important red cell membrane
antigens. Eg. Kell, Duffy and Kidel blood groups.

An antibody screen is performed on the patients serum by incubation

with commercial RBC whose surface antigen expression is known.

Clinical Significance :
Allantibodies may cause hemolytes transfusion reaction or hemolytes
disease of the new born. For a patient who has clinically significant antibody
in the serum, it is important to select blood products.

5) Cross match between patients serum and donor’s RBC.

- Agglutination of donor RBC by patients serum indicates the
pressure of an antibody causing an incompatible cross match.

Clinical significance :
It is necessary to find a compatible cross match, however to avoid
transfusion reaction and ensure the normal survival of transfused red cells.

6) Direct antiglobulin test (Comb’s test)

Used to detect IgG or complement C3B and C3d binding to RBC.
Anti human globulin (anti human IgG) referred to Coombs
reagent causes agglutination of RBC that have surface IgG.
Clinical Significance :
 a) Suspected hemolytic transfusion reaction.
b) Suspected hemolytic disease of new born
c) Suspected autoimmune hemolytic anemia

- ABO blood group was determined on pulp, dentin, and enamel of

35 teeth using adsorption elution technique.
- A bloodstained compress from the examination wound was used
as reference sample.
- 20 were examined with in 6 weeks after examination and 15 were
examined 6-10 months after examination.
- Blood grouping was fairly good in pulp, continued with dentin,
debatable for enamel.

Xing 3 hi x et. Al.

Malaver & Yunis.

- 20 teeth from unidentified bodies
- pulp strongest PCR response
- Dentin & Cementum next.
Metabolism of RBC
- Absence of nu, mitochondria, ribosome and RER,
metabolism prior care absent. Thus tricarboxytes acid cycle of Krebs is
absent in RBC and gen. of ATP is poor.
Glucose is metabolized in RBC and energy in form of ATP is pr
nevertheless.

Rapaport – Leubering cycle operates in RBCs.