Physiology of Blood II

Practicals

• Erythrocyte sedimentation rate

• Count of erythrocytes

• Determination of blood groups ABO

• Determination of Rhesus factor

• Cross-matching test

Erythrocyte sedimentation rate (ESR)

• ESR determination is a simple laboratory test • FW (abbrevation, means Fahreus-Westergren)

• Serum proteins play a crucial role in increased ESR value

• The test measures the distance that erythrocyteshave fallen after one hour in a vertical column of anticoagulated blood under the influence of gravity.

• An elevated value remains a nonspecific finding.

• The most satisfactory method of performing the test was introduced by Westergren in 1921• 4 minute test - rapid

• Wintrobe method – narrow tubes used

ESR - principle

Gra

vitation

alfo

rce

Reference Ranges for the ESR

in Healthy Adults

Adults

Upper limit of

reference range

(mm/hr)

Age < 50 years

Men

Women

0 to 15

0 to 20

Age > 50 years

Men

Women

0 to 20

0 to 30

ESR = erythrocyte sedimentation rate.

Information from Bottiger LE, Svedberg CA.

Normal erythrocyte sedimentation rate and

age. Br Med J 1967;2:85-7.

Factors That May Influence ESR

Factors that

increase ESR

Factors that

decrease ESR

Factors with no

clinically

significant effect or

questionable effect

Old age

Female

PregnancyAnemia

Red blood cell

abnormalities

Macrycytosis

Technical factors

Dilutional problem

Increased temperature

of specimen

Tilted ESR tube

Elevated fibrinogen level

Infection

Inflammation

Malignancy

Extreme leukocytosis

Polycythemia

Red blood cell

abnormalitiesSpherocytosis

Acanthocytosis

Microcytosis

Technical factors

Dilutional problem

Inadequate mixing

Clotting of blood

sampleShort ESR tube

Vibration during testing

Protein

abnormalitiesHypofibrinogenemia

Hypogammaglobulinemia

Dysproteinemia with

hyperviscosity state

Obesity

Body temperature

Recent meal

Aspirin

NSAIDs

NSAIDs = nonsteroidal anti-inflammatory drugs;

Age (years) + 10 (if women)

ESR [mm/hrs]=

2

ESR – how does it look

Red blood cells

• The largest group of blood elements• Transport of gases, especially oxygen

• pH regulation

• Shape biconcave

• Normal count• Females 3.8 – 4.8 x 1012 /L of blood

• Males 4.5 – 5.3 x 1012 /L of blood

V = 85 ± 10 fl

S = 140 mm2

Red blood cell count

• Take 25 ml of blood from finger tip

• Dissolve in small flask with 4.975 ml of Hayem solution• Hayem solution

• Sodium chloride – preserves osmolality

• Sodium sulphate – prevents aggregation of RBC

• Mercuric chloride – antibacterial and antifungal substance

• Water – solvent

• Mix thoroughly

• Put into Burker chamber

Red blood cell count – Burker chamber

Red blood cell count – Burker chamber

• count in 20 rectangles

• the obtained number devide by 100

• subsequently multiply by 1012, which is the result of RBC in 1 L of blood

Result

• RBC count• high altitude

• newborns

• excessive sweating

• hemoconcentration conditions (vomiting, diarrhea)

• chronic hypoxia (congenital heart disease, emphysema, pulmonary obstruction disease)

• polycythemia vera

• ↓ RBC count• pregnancy

• children (when compared to adults)

• women (when compared to men)

• anemias due to different reasons (deficit of Fe, vit B12, folic acid, etc...)

• relative anemia in conditions producing hemodilution (eg. Pituitary tumors and ADH secretion)

↓

Blood groups - introduction

Antigen (Ag)● substance able to start an immune reaction in

organism

●Agglutinogen● antigen on the surface of erythrocyte membrane

●Aglutinin (Ab)● Antibody against agglutinogens

Antigens of blood groups

Glycans/oligosaccharides

proteins GPI - associated

glycosylphosphatidylinositol

Important surface antigens

●29 blood groups●ABO system●Macacus rhesus monkey system – (Rh system)

Significance●theoretical - ?●clinical● transfusion

● gravidity

N° Common name Official abbreviation Epitope or carrier, notes

001 ABO ABOCarbohydrate (N-Acetylgalactosamine, galactose). A, B and H antigens mainly elicit IgM antibody reactions, although anti-H is very rare, see the Hh antigen system (Bombay phenotype, ISBT #18).

002 MNS MNS GPA / GPB (glycophorins A and B). Main antigens M, N, S, s.

003 P P1 Glycolipid.

004 Rhesus RH Protein. C, c, D, E, e antigens (there is no "d" antigen; lowercase "d" indicates the absence of D).

005 Lutheran LU Protein (member of the immunoglobulin superfamily). Set of 21 antigens.

006 Kell KEL Glycoprotein. K1 can cause hemolytic disease of the newborn (anti-Kell), which can be severe.

007 Lewis LE Carbohydrate (fucose residue). Main antigens Lea and Leb - associated with tissue ABH antigen secretion.

008 Duffy FYProtein (chemokine receptor). Main antigens Fya and Fyb. Individuals lacking Duffy antigens altogether are immune to malaria caused by Plasmodium vivax and Plasmodium knowlesi.

009 Kidd JK Protein (urea transporter). Main antigens Jka and Jkb.

010 Diego DIGlycoprotein (band 3, AE 1, or anion exchange). Positive blood is found only among East Asians and Native Americans.

011 Yt or Cartwright YT Protein (AChE, acetylcholinesterase).

012 XG XG Glycoprotein.

013 Scianna SC Glycoprotein.

014 Dombrock DO Glycoprotein (fixed to cell membrane by GPI, or glycosyl-phosphatidyl-inositol).

015 Colton CO Aquaporin 1. Main antigens Co(a) and Co(b).

016Landsteiner-

WienerLW Protein (member of the immunoglobulin superfamily).

017 Chido/Rodgers CH/RG C4A C4B (complement fractions).

018 Hh H Carbohydrate (fucose residue).

019 Kx XK Glycoprotein.

020 Gerbich GE GPC / GPD (Glycophorins C and D).

021 Cromer CROM Glycoprotein (DAF or CD55, regulates complement fractions C3 and C5, attached to the membrane by GPI).

022 Knops KN Glycoprotein (CR1 or CD35, immune complex receptor).

023 Indian IN Glycoprotein (CD44 adhesion function?).

024 Ok OK Glycoprotein (CD147).

025 Raph MER2 Transmembrane glycoprotein.

026 JMH JMH Protein (fixed to cell membrane by GPI).

027 Ii I Branched (I) / unbranched (i) polysaccharide.

028 Globoside P Glycolipid.

ABO system

Genetics• ABO locus

• 9q chromosome

•(17 kb, 7 exons, 7th is the largest, 6th contains deletion)

3 alels: A, B, O

A, B code functional glycosyltransferase, which changes H antigen

O codes non-functional glycosyltransferase

4 antigensA, B, AB, A1

●They are determined by the sequence of oligosaccharides/glycoproteins●N-acetylgalactosamine/D-galactose

● universal donor● universal recipient

• watch out for bacteriemia !!!

Where are the antibodies produced?

Agglutination

Coagulation

Bombay phenotype ( Oh)

• 1952 – Bombay, an individual was reported, whose serumagglutinated all blood groups A, B, 0

● Locus FUT 1 was localised on19q●codes fucozyltransferase – the production of H antigenon the erythrocyte surface●homozygots h/h●the production of anti-A, anti-B, anti-H

Rh factor

In 1939 a mother gave 2nd child, who was dead at delivery(birth) and the mother needed transfusion

ABO system was recognised for 40 years by then, and blood group compatibility was the necessity for transfusion

her husband had the same blood group

however, mother died

What had happened?Why did the child die?

Why did the mother die?

At pregnancy, which antibodies are produced?

Why Rh factor? In serum of animals, antibodies were produced after M. rhesus

monkey erythrocytes were injected. It was believed, that these Ab are the same class/specificity as Ab found in mother serum after transfusion of husband erythrocytes.

Rh factor

• It is most important after ABO system

• Rh factor is determined by the presence of D antigen

• Genetics

• genes at chromosome 1p

• 2 genes

• RHD, RHCE

• 49 known antigens

Rh factor

• It does not contain oligosaccharides

• Associated with RhAg

• this complex is responsible for the ammonia and CO2

transport accross the membrane and for the stability of ERY

membrane

• the absence of Rh factor will increase the ERY osmotic

fragility

• Is also responsible for hemolytic disease of the newborn

(HDN)

Duffy system

• First time described in 1950, in a human withhemophilia, who received transfusions repeatidly

• A year later was discovered 2nd antigen in a woman with several children, and the other Agwere discovered 20 years later

• Overall - 6 Ag

• FYA, FYB, FY3, FY4, FY5, FY6

• Of last four Ag, clinical importance has only FY3

• FY null phenotype is often in afroamericans

• Why?

Duffy system

• Genes are coded on chromosome 1q

• Ag = receptor

• Substances excreted during inflammation– cytokines activate

this receptor

• Plasmodium vivax activates this receptor

• Codominant alels FYA and FYB

• 68% FYa and FYb in afroamericans

• resistance to malaria

Hemolytic disease of the newborn

1.ERY of Rh+ child gets into Rh- mother during the first delivery

2.mother creates anti-Rh+ antibodies

3.during the 2nd pregnancy and Rh- child, mother Ab get into the fetus and destroy fetus ERY

• Which antibodies are responsible for the 3rd stage?

IgG !!!

Blood and blood derivates transfusion – side effects

• Determined by immune system

• Acute hemolytic transfusion reactions• intravascular hemolysis

• extravascular hemolysis

• late hemolytic anemia

• Febrile non-hemolytic transfusion reaction• leucocytes incompatibility

• trombocyte incompatibility

• alergic reaction (IgE Ab)

• anaphylaxis (IgA Ab)

• TRALI (Transfusion related acute lung injury)

• TA-GVHD (transfusion associated graft vs. host reaction)

• Not determined by immune system

• hypotermia

• volume overload

• metabolic disorders

• hypercaliemy/hyperpotassemia

• hypocalciemy

• coagulapathies

• infections

• HIV, HBV, HCV, HTLV I. and II., CMV,...

Blood and blood derivates transfusion – side effects

Questions?

• Reading for next practicals: Blood Groups and Red Cell Antigens

• https://www.ncbi.nlm.nih.gov/books/NBK2261/?term=blood%20groups• Chapter 1, 2

• Chapter 3 (until Transfusion reactions)

• Chapter 4, 5, 6, 7 and 9

• If interested: 8 and 11

Go to google.com, insert all key words: Blood groups and red cell antigen

It is the first link through NCBI Bookshelf