Haemat Pracs: WEEK 1

9/2/23

• Duke's test- 3-8mins (normal coagulation time) - earlobe

• Ivy's test- 1-7mins -upper arm

Coagulation factors:

1. Intrinsic coagulation pathway- d/t pregnancy, drugs, inherited, surgery etc. Thus thromboplastin is
formed externally. Factor 1,8,9,11,12, prekalikulin* ,high molecular kininogen. Test: .... (30-40sec)

2. - Factor 7, 10, 5, 2 etc. - a collective factor pathway - Test: Prothrombin Time Test (in seconds- 13-16
s: normal), work with plasma: Parameters of reporting- test, (control, PTI(%) Prothrombin control/....
Raise by an International Sensitivity Index (1.01-1.2)

3. Common pathway: factor 1 - fibrinogen. (Special test): Thrombin Time (8-11 sec).

Urea complex degraded by plasmin in the LIVER. (Plasminogen- active plasmin) into X fragment and
peptide. D-Dimer test (special test): levels raised in smokers, immobilized patients, after surgery, old
age.

• READ ON PT AND APTT

Other tests: LFT, albumin(factor inhibitor test), fibrinogen, FDPs(Fibrin degradation products)- D-Dimer
test,..., complete blood count/PBF/Bone marrow

Haemat Pracs: WEEK 2

13/2/23

1. TOTAL BLOOD COUNT

-EDTA : Purple vaccutainer

-2mls generally

-QC is done first

-Haemolytic machines are used e.g. sysmex, coulter

-Readings are interpreted from a printout:

• WBC, NRBC, NEUT, LYMPH, MONO, FO, BASO, IG

• RBC, HGB, HCT, MCV, MCH, MCHC, RDW(Reticulocyte Distribution Width),

• PLT, PDW, MPV (Mean Platelet Volume) , P-LCR, PCT

• TRF, LFR, MFR, HFR, RET-He, RET

Buy/Download: Hematology Technical Manual

-Manual Hb Devices:

• Tallquist: filter papers& a colour chat

• Hb meter: match colours to get level of Hb

• sahli's method

-WBC manual devices

• Improved neubauer chamber

*There is a 4×4 square on a glass slide seen under a microscope. It comes with a white blood cell diluting
pipette. Pipette blood and add diluting fluid. Leave for 5 mins for reaction. (The diluting fluid lyses all
RBC and stains the WBC to appear as dark particles). After diluting, drop the solution onto theglass slide
and observe under a microscope. Count the no. Of WBC in the 4 corner squares and get the total to give
you the total WBC count. (For rbc&platelet, check the center square)

•
*In the exam they can ask for the differential diagnosis

E.g. leukocytosis: leukemia etc

Low platelets: thrombocytopenia

16/2/2023

EDTA samples

-Malaria-Thick smear

-FNA- Thin smear

-PBF-Thin smear

Types of slides: 1. 2. (Has a portion left for labelling)

- CSF: Centrifuge then make a thick smear of the sediment on a glass slide

A. THIN SMEAR-

B. FNA-

C. SQUASH-

D. STRAIGHT/PUSH SMEAR- Like a PBF(thin smear)

2. ESR

-REDUCED

• <4mm/h

• CONDITIONS:

• polycythemia

• sickle cell anemia

-INCREASED

• CONDITIONS:
• Tuberculosis

• Multiple myeloma

• severe anemia

• Bacterial infections

• Leukemia

• etc.

-Bone marrow needles: Kilma& Salah

17/02/23

RETICULOCYTE COUNT:

Formula:

{(No. of reticulocyte÷1000)×100}%

Increased:

1. Haemolytic anemia

2. Patients on hematunics

Reduced:

1. Any condition suppressing BM e.g. HIV, Aplastic anemia

2. Patients on cytotoxic drugs

SICKLING TEST

-Screening test for sickle cell anemia

-Usual Methods of testing for SCA:

• PBF

• Sickling test

• Hb electrophoresis
-Principle: CELLS ONLY SICKLE IN ABSENCE OF OXYGEN. THE WORKING SOLUTIONS ARE OXYGEN
REDUCING AGENTS AND THUS THE CELLS DEFORM INTO THE OBSERVED SHAPE WHEN THE SOLITION IS
ADDED.

-Procedure:

Add drop of blood on glass slide. Add 3 drops of 3% working solution. Put a cover slip. Seal with paraffin
wax/Vaseline. Incubate for 30mins @37°C. Observe under microscope on ×10 or ×40.

-Morphologies of sickle cell ("sharp-ended"):

• Acanthocytes/Schistocytes (but reported as a sickle cell)

• Sickle cell

• Helmet cell

-Report: "Sickling test Positive"

-Errors in the test:

• Poikilocytosis (e.g. spherocytes may look like sickle cells)

• Use of conc. soln: Water is drawn out of the cells by osmosis giving them a morphology that may be
mistaken to be sickle cell

• Short incubation period: not enough time for oxygen to be depleted

• Poor sealing of preparation: Leakage of oxygen into the sample

• Expired reagents

HB ELECTROPHORESIS

- Confirmatory test for sickle cell disease

- Principle: HB IS NEGATIVELY CHARGED AND WHEN EXPOSED TO AN ELECTRIC CURRENT, THEY'LL FLOW
IN THE DIRECTION OF THE CURRENT

- METHODS:

1. Cellulose Acetate Paper Electrophoresis (CAPE)

2. Agar gel electrophoresis✔

3. Capillary electrophoresis✔

4. Starch block electrophoresis

1. CAPE
-Electricity, electrophoresis tank, cellulose acetate paper (CAP), blood sample (mainly EDTA), buffer (pH
6-9)

-Separation of Hb such that Hb with more iron go a farther distance on the CAP.

2. ...

3. CAPILLARY ELECTROPHORESIS

Haemat Pracs: WEEK 3

Monday 20/2/2022

SUMMARY:

A. BLEEDING PROCEDURE

B. SCREENING

C. STORAGE

D. COMPATIBILITY TESTING

E. SEPARATION OF BLOOD PRODUCTS

A. BLEEDING PROCEDURE:

************************

-Bleed into the primary/main bag (where all the other bags originate from)

-Anticoagulant/preservative: CPDA (Citrate-anticoagulant, Phosphate- maintains blood pH, Dextrose-

source of energy for the cellular elements of blood, Adenine- inc. survival rate of RBC by providing ATP)-
Blood preserved for 35 days

-Use the pilot tube

-The tube and bag have an identification number (donor's/ unit number)
-Guage 19 needle

-Angle 30 then along the vein

-Secure needle using strapping

-Donor presses ball on the hand to facilitate blood flow

-Blood bag capacity 450-500ml

-once full, release tourniquet

-apply pressure with cotton wool and secure

-Donor to lie in the same position for 5-10mins to assess their condition before leaving

-Donors are given fluids to replace the lost one e.g. Soda, milk, water

B. SCREENING:

-For transfusion transmitted infections (TTIs)- HIV 1&2, Hep B&C, Syphilis, Malaria

C. STORAGE:

-Whole blood Refrigirated @ 2-8°C for 35 days (Reasons: Slow down cellular metabolism, Reduce
bacterial growth)

D. COMPATIBILITY TESTING:

E. SEPARATION OF BLOOD PRODUCTS:

-Blood bag sets: (Named according to the number of transfer bags in the set):

a. Single bag-only primary bag (For whole blood transfusion)

Multiple blood bag sets: (For separation of blood products)

b. Double bag-Primary bag+1 transfer bag

c. Triple bag- Primary bag+ 2 transfer bags

d. Quadruple bag- Primary bag+ 3 transfer bags

-The primary bag has the:

• Donor number

• collection date and expiry date

• ABO type and the Rhesus type- Major blood group systems (Just a mention: Minor/rare blood group
systems: Kell, Duffy, Kidd, Lewis etc.)

-Blood products: Fresh Frozen Plasma, Platelet concentrate, Platelet Rich Plasma, Packed RBC,
Cryoprecipitate, WBC concentrate.

-Methods of making blood products: Automation (Apheresis technology), Manual( centrifuge:

(Parameters change: Time, speed, temperature):

1. Fresh Frozen Plasma:

INDICATION

-Correct bleeding disorders due to coagulation factors deficiency or coagulopathy.

-Clinical conditions:

GENETIC/HEREDITARY

a. Hemophilia: A (Classical)- factor 8 deficient, B (Christmas disease)- factor 9 deficient

b. *

ACQUIRED

a. Liver disease: affects synthesis of coagulation factors

b. DIC: High consumption thus deficiency of coagulation factors

c. Vit K. deficiency: affects synthesis of coagulation factors

d. Renal disease: Coagulation factors filtered out

METHOD

-Prepare blood products within 8hrs of collection.

-Use fresh blood to preserve the labile factors: 5&8.

-4200rev/min, 9min, 4°C... From the primary bag, use a plasma extractor then break the tube and use a
tube sealer)

STORAGE

-*Conditions: Freeze(to preserve labile factors)

-Life span: 1 year

-ABO type, screening info. included

2. PLATELET CONCENTRATE

INDICATION:
-Correct bleeding disorders due to low platelet count or thrombocytopenia

-Platetlet reference range: 150-450

METHOD:

(No need to know)... According to lec

STORAGE:

- Placed on a platelet agitator (To ensure the platelets are continuously mixed thus prevent platelets
from clumping) AT ROOM TEMP.

- Life span: 5days

3. CRYOPRECIPITATE

INDICATION

Correct Classical haemophilia, VW Blood disease, Hypofribrinoginemia

-Contains: Factor 8, Bon something*, Factor 1, Factor 13

-Prepared from Fresh Frozen Plasma

-Has all the coagulants except the ones targeted by the anticoagulant (Calcium ions(factor 4))

METHOD:

-Those 4 coagulation factors are high molecular weight proteins which normally form a precipitate when
FFP is slowly thawed at 4°C for 24hrs. Centrifuged and excess plasma discarded leaving about 15-20mls
to resuspennd the precipitate

-Life span 1yr

-temp: same as FFP

4. PACKED RBC

INDICATION:

-INCREASE oxygen carrying capacity/RBC mass in anemic patients

-Sickle cell crisis

METHOD:

Whole blood centrifuged then plasma is...

STORAGE:

-like whole blood

-Preservative: Saline Adenine Glucose Manittol -for 42days