LAB EX.

# 1LAB SAFETY & QC OF RGTS & EQPTS  Proper handling & disposal of all specimens tested Layers:
 1˚ focus of safety: biohazardous materials  Immediately clean any spillage w/ suitable disinfectant w/ weak Blood Products Temp storage
 biohazard materials – all body fluids & tissue in the lab & the Hypochlorite soln. WB 1-6˚C
reagents used that are of human origin  Properly decontaminate all waste before disposal RBC’s 1-6˚C
 Factors affecting the accuracy of the test:: FFP -18 to -30˚C serum plasma
 Properly label all hazardous waste as BIOHAZARD
Platelets 22-24˚C
1. Reagents’ stability  Separate containers for hazardous & non-hazardous waste Washed RBC 1-6˚C buffy coat
2. Equipment Waste management Filtered RBC 20-24˚C
 Quality Assurance Program – evaluate test procedures being prfm a. Hazardous waste Cryoprecipitate -30˚ C
& correct prblm that arise in the test or w/ the eqpts packed RBC clot bld
 Infectious waste: any material/equipment w/c used to contain Cryosupernate -30˚C
– method of ensuring error-free performance ****Demonstrations of the bag refer to the pics of Ria. *buffy coat:
hazardous elements (test tube, pipet,…)
– comprehensive program that monitor & evaluate all top layer: platelets
 Sharp objects: needles, syringes,… ***** Notes fr Lab tour:
aspects of the performance 2nd layer : WBC lymphocytes
 Pathological wastes: actual specimens (B, serum,…) Bld prd Memory # Temp Speed Time
– encompass the categories of personnel, policies & (rpm) monocytes
 Chemical wastes: any substance or soln. w/c is flammable,
procedures, & techniques Packed 1 5˚C 3210 7 mins granulocytes
corrosive; irritating/strongly sensitizing
– incl testing kit, ( + )& ( - ) controls RBC  Factors that affect the degree of packed RBC:
 Disposal: chemically disinfect or autoclave; must undergo pre-
 Food & Drugs Asso. – regulating agency in the US Plt conc 1. speed & time of centrifugation
treatment process before incineration &/or sanitary landfill burial a. light spin 2 22˚C 1940 5mins
 American Asso. of Blood Bank – accrediting agency in US 2. radius of centrifuge
in adequately secured lot b. heavy 3 22˚C 3210 7 mins
 Dep. of Health thru the Bureau of Lab & Research – regulating 3. height of the blood column
b. Non-hazardous waste spin
agency in Phil.  Hemolyzed samples free RBC Hb that mask Ab induced hemolysis
 Not known to pose substantial hazards to human health or PRP 4 22˚C 1940 5m ins
 Records of Daily Reagent Controls:  washed RBC – used in serologic test for IM, ASO test; Blood bank:
environment (paper, office supplies) Plasma 5 5˚C 4060 3 mins
1. Date of testing Cryo ppt 6 (1-6˚C) 4100 5 mins blood typing, compatibility testing
 Disposal: use sanitary landfill burial, composting of
2. Source of Reagent Used 5˚C – act as in vitro Ag
biodegradable waste, recycling scheme or ordinary disposals
3. Expiration date  Centrifuge door must be open after every use until the moisture  Purpose of washing RBC:
Color coding of waste containers:
4. Lot number of reagents has evaporated 1. remove plasma or serum to prevent formation of fibrin clots
 Red – sharps
5. Identification of person performing testing  Separation stand – use to separate plasma in an open system 2. remove serum factors that inhibit complement reactivity
 Yellow – infectious waste
 Reagents : bag 3. removes soluble Ag-Ab complexes that compete w/ the target
 Green – non-infectious waste
1. ABO Typing Sera  Open system – life span: 24hrs cell for complement
 Black – non-infectious waste (dry)
2. Rh Typing Serum  Dia Med-ID Micro Typing System – use in crossmatchin. 4. removes unbound globulin by dilution
 Yellow w/ black band – chemical waste
3. Anti-human Globulin Serum – incubation: 15 mins  Procedure of washing:
Decontamination:
4. 22% Albumin – centrifugation : 10 mins 1. Add NSS to the cells in the EDTA tube, mix w/ paraffin
 Chemical – e.g. 0.1 – 0.5% Hypochlorite
5. 0.9/0.85% NSS 2. centrifuge for 1 min @ 3400 rpm, remove the NSS layer and
 Autoclave – 121°C for 15 mins.
 Materials: LAB EX # 3 DISTINGUISHING OF SERUM & PLASMA PREP OF discard it into the beaker containing 10% sodium hypochlorite
 Dry heat sterilization – 170°C for 2 hrs. 2% RBC SUSP’N
1. 12x 75mm Test Tube (chemically clean & dry) 3. repeat steps 1-2 again and proceed to making a cell suspension
 Equipments: Plasma Serum  2% cell suspension – gives the best result
LAB EX # 2A VISIT TO THE BLOOD BANK
1. Centrifuge -liq portion of a - fluid portion of a – adv:
CEBU VELEZ GENERAL HOSPITAL
2. Water Bath/ Hot block blood sample blood sample that 1. more sensitive for agglutination
 category: Blood Bank/ Center Category B Hospital base collected w/ has clotted
3. Rh Viewing Box 2. avoids zonal phenomenon
 services: anticglnt (chelates – serum to cell ratio: 2:1
 Emergency eqpt; 1. Recruitment of Voluntary Blood Donors Ca) – inc the serum to rbc ratio inc the demonstration of weakly
1. Fire Extinguisher 2. Health Education & Counseling Clotting factors 1,5,8,11,12,13 4,7,9,10,11,12,
reactive Ab
2. Fire blankets 3. Donor Screening & Selection present
Clotting factor Ca Fibrinogen  fibrin – it is best to use the weakest cell suspension that can observe
3. First Aid Kit 4. Blood Collection
absent easily for agglutination
4. safety shower 5. Blood Screening & Testing for Blood Transmittable Diseases
5. Eye Wash Station Adv to serum only:  Agglutination – dep on a minimal # of Ab molecule/ rbc
6. Provision of Whole blood, RBC & all blood products 1. doesn’t contain fibrinogen
 Classes of Fire:  Procedure of making a cell suspension:
7. Storage of Blood & Blood Products 2. Ab demonstratable
1. Class A – combustible 1. After the removal of plasma, wash the RBC w/ 0.9% NSS for 2X.
8. Issuance, Transport & Distribution of Whole blood, RBC & all 3. contains Ca needed in most test
2. Class B – flammable liquids 2. Last wash is the most critical step. Remove all NSS to prevent dil
blood products Procedure: 1. collect blood 1. collect blood
3. Class C - electrical effect that leads to false ( - )
9. Compatibility testing 2. use lavender top 2. use red top
 Factors that need checking in the lab: (EDTA) (plain) 3. 4.9 ml NSS in a test tube + 0.1ml washed RBC
10. Investigation of Transfusion reactions
3. centifude for 5 3. clot bld for 5-7 4. Mix using paraffin to prevent contamination & to protect the
1. Ref temp 11. Resolution of Incompatible Crossmatches
min @ 3400rpm mins health worker
2. centrifuge timer, & balance  Donor Screening Test: 4. remove plasma 4. centrifuge for 5  Formula used: C1V1=C2V2
 PRACTICE IS THE KEY TO ACCURACY a. ABO & Rh typing 5. proceed to cell mins @ 3400 rpm
 Handwashing – most impt way to prevent bacterial or viral b. Ab screening suspension
LAB EX # 3 GRADING AGGLUTINATION RXN
contamination c. Serologic screening (syphilis)
 Agglutination – the clumping of particles with Ag on their surface
 ALWAYS FOLLOW MANUFACTURER’S INSTRUCTION d. Viral testing ( HIV, HsB Ag, HCV, Malarial smear) such as erythrocytes, by antibody (Ab) molecules that form bridges
Safety measures in BB e. Transfusion-transmitted viruses between antigenic determinants of adjacent cells
 Physician available at all times & emergency facilities  Donor Selection Process: – endpoint for most in vitro tests involving erythrocyte Ags and
 Report & properly document all accidents a. Pre-donating Process blood group Abs
 Regular testing of all staff for HBV b. Donor Screening
 Immunization of staff against HBV c. Post- donation process
 – can be observed either through direct techniques such as ABO 1. 1 drop serum + 1 drop RBC susp’n in a 12x75 mm test tube LAB EX 5A ABO BLOOD GRP – SLIDE/ TILE METHOD + 1 drop 4% RBC susp’n 1 drop px serum +
testing & Rh typing or Indirect techniques such as Antiglobulin test/ 2. Mix & centrifuge for 1 min @ 1000 rpm ( Serofige)  Karl Landsteiner – discovered the ABO blood grp in 1900 1 drop Anti –B 1 drop B cells
Ab screening 3. Dislodge RBC clumps to grade agglutination. – grps: A, B, & O Centrifuge for 15 sec @ 3400 rpm and check for agglutination
 2 Stages of Agglutination: STRENGTH – his pupils, Starie & von Descatello, discv AB * 4% RBC susp’n: (final vol = 5 mL)
OF GRADE SCORE APPEARANCE 4.8mL NSS + 0.2 mL washed RBC
1. Sensitization – first phase where there is physical REACTION  ABO blood grpng – is 1 of the 1˚ test perform in the blood bank
attachment of Ab to Ags on the RBC membrane when Ab Reagents:
. 4+ “COMPLETE” 12 A single agglutinate. No free RBCs  LAW: each individual produces Ab corresponding gto the Ag
combines with Ag, sensitizing the Ag. detected A1 Cells B Cells
absent on the surface of the RBC
–a reversible interaction when antibodies combine rapidly with 3+ 3+ 10 Strong reaction. A number of large Source Human RBC
Ag-nic particles agglutinates  2 types of test:
– affected by the equilibrium constant or affinity constant 2+ 2+ 8 Large agglutinates in a set of smaller 1. Forward typing – use known Ab to det the presence of Ag
clumps, no free RBCs
– the larger the equilibrium constant the greater the rate of present on the surface of the RBC
1+ 1+ 5 Many small agglutinates & a
asso. & the slower the rate of dissociation background of free RBCs – suitable for newborn & infants less than 6 months old ( Ab are
– factors / physical conditions affecting physical attachment: Results:
½+ or (-) +/- 3 Weak granularity in the suspension. A synthesize starting in 6th month & are well dev on the 5th or 6th
a. pH Bld Grp Anti-A Anti-B A1 cells B cells
Macro few macroscopic agglutinates but yr)
b. ionic strength - concentration of salt in the reaction medium numerous agglutinates A + - - +
2. Reverse typing – use known A & B cells to det. the presence of B - + + -
– lowering/reducing ionic strength of a reaction medium, microscopically
enhances Ab uptake Trace or (+) Micro 2 Appears negative macroscopically. A Ab in the serum. O - - + +
– enhancement agents: Micro few agglutinates of 6-8 RBC in most – can’t be used for newborn babies , and infants less than 6 AB + + - -
 low ionic strength saline (LISS) fields months old
0 0 0 An even RBC suspension. No
 Polybrene – uses:
agglutinates detected
c. temperature: a. to validate the results fr the Forward typing
 IgM – 5-24˚C Ways of dislodging RBC clumps:
b. to check the reactivity of the anti-sera
 IgG – 37˚C a. wiggle
2. Lattice Formtion - establishment of crosslinks between  Common charc of Ab of the ABO bld grp sys:
b. rub bet hands
sensitized particles such as RBC and Ab resulting in 1. Agglutination of saline suspended RBC (they are IgM class Ab)
c. tap w/ finger gently
aggregation 2. Reactivity temp is @ 4-22˚C
d. flick
– slower 3. Produces hemolysis when binds w/ complement
– crosslinking is affected by zeta potential, Ab type, proteolytic 4. Tilt to see the clumps
4. Inactivation of IgM anti-A & ant-B w/ 2-mercaptoethanol (2-ME)
enzyme, Ag dosage 5. use a mx to confirm
IgM IgG
 Zoning Phenomenon
Optimum temp 4-22˚C 37˚C
Pro-zoning – excess Ab  false ( - ) Optimum Saline Albumin
Post-zoning– excess Ag  false ( - ) medium
 METHODS OF ENHANCING AGGLUTINATION Classification Saline-agglutinating Incomplete or
1. CENTRIFUGATION or complete or univalent or
2. TREATMENT with proteolytic enzymes (eg: papain, ficin, bivalent coating or blocking
Bromelin) which also eliminates the Ag-binding sites for other Passes thru the No yes
Antibodies such as Anti-M, Anti-S, Anti-Fya, anti-Fyb. placenta
3. The use of COLLOIDS –Bovine Albumin 22% protein
concentration , pH 7.2 with 0.1% sodium azide as preservative  Rule: Serum 1st before RBC
and acacia
4. Addition of anti-human globulin (AHG) reagent  Procedure :
5. The use of LISS Forward Typing Reverse Typing
6. Polybrene 1 drop Anti – A 1 drop A cells
7. POLYETHYLENE GLYCOL (PEG) – the more effective in + 1 drop 2% RBC susp’n 1 drop px serum +
detecting weak antibodies than LISS manual polybrene 1 drop Anti –B 1 drop B cells
 Psuedoagglutination – erythrocytes microscopically resemble rolls Mix w/ an applicator stick and rack for about 2 mins (maximum time
of stacked of coins, rouleaux formation. to observe for agglutination
– to differentiate from true agglutination;
1. examine usin mx Reagents:
2. use saline replacement technique (never done before initial Anti-A Anti-B
testing protocol  dil. effect  false ( - ) Source Monoclonal Ab ( mouse)
Grouping Highly specific IgM
– factors enhancing rouleaux formation:
Color blue yellow
 High Protein Reagent (Anti-D)
Dye none Acroflavin
 Rouleaux formation may occur rapidly, particularly on
warm slide resulting to partial evaporation then drying.
This is due to increase of protein concentration in the 5B ABO BLOOD GRP – TUBE METHOD
reaction mixture. - more sensitive bec:
 Abnormal increase of concentration of plasma proteins a. the use of 4% RBC susp’n
like increased fibrinogen levels b. centrifugation – enhances agglutination
 Inverted Albumin-Globulin ratio (N.R. : 2:1) - tubes used
 Transfusion of a high molecular weight IV solution
 RBC susp’n – 12x75mm
(Plasma Expander)
 Cord Blood samples from Newborn infants which contains  rxn tube – 10x75mm
Wharton’s Jelly  Procedure:
 Infx with increased ESR Forward Typing Reverse Typing
 Procedure: TRUE AGGLUTINATION 1 drop Anti – A 1 drop A cells