B.H.

Y
HOSPITAL
LABORATORY
MANUAL
BLOOD GROUP SYSTEM
 ABO BLOOD GROUPING:
PRINCIPLE:
ABO antibodies are of IgM class and react at room temperature .Incubation at warm temperatures may
cause a false negative reaction.

ABO FORWARD TYPING:

This test is used to detect the presence of absence of A and/or B antigens on individuals red blood cells.

PROCEDURE:
 Caution :
Never place any specimen or reagents in an unlabeled tube .

 Write patient name and lab no on all three tubes and suspension tube .
 Check patient’s name and lab no on EDTA tube (purple top ).
Place tip of the pipette at the bottom of sample and aspiratae 200 µl of cells and place in cell
suspension tube.
Fill the tube containing cells three.fourth full with saline using enough force to thoroughly
resuspend.
 Place cell suspension in the centrifuge .spin for mins at 1500 rpm.
 Wash cells three times with normal saline .
 Label three tubes with patients initials at the top and lab no at the top of each tube .
 Place in a rack as indicated below:

SAMPLE Pt. RBCs Anti -A Anti -B Anti –D

Suspension
 Decant the saline from the cell suspension completely by inverting the tube over the waste
bucket , allowing all saline to drain out , shake the inverted tube 3 time , do not shake tube
while saline is draining out .
 Add enough saline to the patients cells to obtain or 4-6% cell suspension.
 Add one drop of reagent antiserum in labeled tube ( anti -A in A tube , anti -B in B tube and anti
–D in D Tube ).
 Add 200 µl of patient cells to each tube .
  Mix tube thoroughly by gentle shaking .
 Spin all tubes for two minutes at 1500 rpm.
 Read and immediately record reaction .
 Shake tube gently to dislodge cells .

ABO REVERSE TYPING:

PROCEDURE:
 Centrifuge Red top tube in order to separate patient serum.
 Label two tube with patient initial and lab no .
 Add 200 µl of serum in tube 1 and similarly in second tube.
 Add 200 µl prepared A1 Cells in tube 1 and 200 µl prepared B cells in tube 2 .
 Mix well and centrifuge both tubes at 1500 rpm for 2 min .
 Remove the tubes and resuspend cells examine macroscopically for agglutination and if
negative, microscopically .
 Record the reaction and interpret result .

FORWARD REVERSE BLOOD

GROUP

ANTI- A ANTI - B ANTI -D A CELLS B CELLS

O O + + + “O” POSITIVE
+ O + O + “A” POSITIVE
O + + + O “B” POSITIVE
+ + O O O “AB”POSITIV
E
O O O + + “O”NEGATIVE
+ O O O + “A”
NEGATIVE
O + O O + “B”
NEGATIVE
If
patient is Rh negative; WEAK D .Test must be performed.
 WEAK D TEST:
PRINCIPLE:
Some red cells possess the D antigen but it is expressed so weakly that the cells are not
agglutinated directly by anti D sera. An indirect antiglobulin test is necessary to identify patients
with weak D (formerly known as Du) phenotype .

 Weak D testing is done on all prenatal patients.

 Weak D testing is also done on Rh negative donors to ensure they D-negative.

SAMPLE:

Washed cell suspension prepared from EDTA anticoagulated tube is used.

SUPPLIES:

 37 degree incubator
 Coombs reagent
 Coombs control cell
 Anti D reagent

PROCEDURE:

 If tube containing anti D is negative, set up a tube labeled with patients initials and D
control.
 Place one drop of Rh control in the tube followed by 200µl of patients washed RBC
suspension .Spin, read the D control tube.
 Place both the negative D tube and D control in incubator for 30 -40 mins.
 After 30 mins, remove the D tube and D control, wash three times with saline .
 After last wash add two drops of AHG to each tube .Mix well and and centrifuge for 2
mins.
 Gently resuspend cell button, looking for agglutination.
 If negative macroscopically, observe microscopically.
INTERPRETATION:

D TYPING WEAK D
Anti D D D control
O 3+ O POSITIVE
O O O NEGATIVE
O 3+ 3+ INVALID
 COOMBS DIRECT ANTIGLOBULIN
TEST:
PRINCIPLE:
Red cells coated with complement or IgG antibodies do not agglutinate directly when
centrifuged. These cells are said to be sensitized with IgG or complement. In order for
agglutination to occur an additional antibody, which reacts with Fc portion of IgG antibody,
must be added to the system. This will form a bridge between the antibodies or complement
coating red cells, causing agglutination.

 The DAT is used to detect antibodies that are stuck to the surface of rbcs.
 These antibodies sometimes destroy RBCs.

PROCEDURE:

 Prepare a 5% suspension of RBCs to be tested in isotonic saline.

 Label three tubes as T, PC and NC.
 In the tube labeled as T (test) take 200 µl of 5% cell suspension.
 In the test tube labeled as PC (positive control), take 1 drop of anti D sera and 200µl of
Rh +ve pooled cells.
 Add two drops of AHG to each of the tubes.
 Mix well and centrifuge for 1 min at 1500rpm.
 Resuspend the cells by gentle agitation and examine macro and microscopically for
agglutination.

INDIRECT ANTIGLOBULIN TEST:

 IAT is used to detect in vitro antibody –antigen reaction .
 Used to detect very low concentrations of antibodies present in a patient’s serum prior
to a blood transfusion.
 This test is used to screen pregnant women for antibodies that may cause hemolytic
disease of newborn.

PROCEDURE:

 Label three tubes as T, PC, and NC.

 In the tube labeled as T (test), take 200 µl of test serum.
  In the tube labeled as PC (Positive control) take 1 drop of anti D serum.
 In the tube labeled as NC (Negative control),take 1 drop of normal saline.
 Add 200 µl of 5% cell suspension of pooled ‘O’ Rh +ve cells in each tube.
 Incubate all the tubes at 37degree for 60 minutes.
 Wash cells 3 times with N/S .
 Add 2 drops of AHG to each tube.
 Keep for 5 mins and centrifuge at 1500 rpm for 1 min.
 Resuspend the cells and examine for agglutination microscopically and macroscopically.

INTERPRETATION:

If the blood coagulates, it can be concluded that the patient’s RBCs have been bound by his
own immunoglobulins.
 Rh ANTIBODY SCREENING
SCOPE AND APPLICATION:
This procedure applies to all testing that requires antibody screening, including donor units and
prenatal specimens.

MATERIALS REQUIRED:

Equipment:

 Refrigerator to store samples & reagents at 2-8 0 C.

 Deep freezer to store enzyme, albumin & AHG reagent.
 Table top centrifuge.
 Automated cell washer.
 Microscope.
 Water bath.

REAGENTS:

 Group O pooled cells  22% Bovine albumin Antihuman globulin reagent  IgG
sensitized control cells 0.9% saline Distilled water
 Enzymes

SPECIMEN:

Clotted blood sample of donor.

PRINCIPLE:
The antibody screen test is used to detect unexpected antibodies. In
this test, pooled O cells are combined with serum under investigation.
The addition enhancer medium enzyme/ albumin helps to promote the
interaction of red cells and antibodies allowing antigen-antibody
reactions to occur.
PROCEDURE
Antibody screen:

 Label tubes with donor/ patient and test identification. Test tube (number)
 Add 2 drops of serum to each tube.
 Add 1 drop of enzyme to the tube labelled ‘enzyme’. To each of the tube labelled
‘saline’, ‘enzyme’ and ‘ICT’, add 1 drop of 5% pooled O red cell suspension.
 Incubate the tube labelled ‘saline’ for 1hr. at room temperature.
 Place the tubes labelled ‘enzyme’, ‘albumin’ and ‘ICT’ into the incubator at 370 C for 1hr.
 After 1 hr, examine the tube labelled ‘saline’ for agglutination.
 Remove the tubes labelled ‘enzyme’, ‘albumin’ and ‘ICT’ from the incubator and add 1
drop of albumin to the tube labelled ‘albumin’.
 Again place tubes labelled ‘albumin’ and ‘ICT’ to the incubator for ½ hr. more.
 After ½ hr. incubation, tubes are from the incubator and tube labelled ‘albumin’ are
examined for agglutination.
 Positive (IgG serum) control and Negative (AB donor serum) control
 Add pooled cell.

RESULTS:

 Gently re-suspend the red cell button and examine for agglutination.
 Examine all negative results microscopically.
 Proceed to perform indirect anti-globulin test on tube labelled ‘ICT’.
 Wash the cells 3 times with saline. Decant completely after last wash.
 Add 2 drops of AHG reagent to the cell button.
 After 6-8 minutes, read and record the results.
 Add 1 drop of IgG sensitized cells to all negative results. This shows a positive result.

INTERPRETATION:
Haemolysis or agglutination in any test may indicate the presence of an unexpected antibody.
The absence of agglutination in all tests is a negative test. After addition of IgG sensitized cells
to a negative test, the presence of agglutination indicates that the AHG added was capable of
reacting and that the negative result is valid.
 RH ANTIBODY TITRE:
PRINCIPLE:
Titration is a semi quantitative means of measuring the amount of antibody in a patient’s
serum. Serial dilutions of the antibody are reacted with a constant volume of the specific rbcs
and the results are expressed as the reciprocal of the highest dilution in which agglutination is
observed.

The usual applications of titration studies are:

 Estimating the antibody activity of an alloimmunized pregnant female.

 Attempting to determine if there is any specificity to an auto antibody.
 Characterizing antibodies that may be high titer, low avidity antibodies.

SAMPLE:

Blood should be drawn aseptically. Serum should be used. Store sample at 2-8 degree until
testing can be done.

If possible test the current sample in parallel with the most recent previously submitted sample
from the current gestation.

MATERIALS:

 Anti human IgG

 Isotonic saline
 Volumetric pipettes
 Red cells group O reagent cells, 2% suspension.
PROCEDURE:

 Label 10 tubes according to the serial dilution: 1, 2, 4, 8, 16, 32, 64, 128, 256, 512 and
patient identification.

Save the last tube in case a higher dilution must be prepared.

 Add 100 µl of saline to tubes ‘2’ through ‘512’ .No saline in tube ‘1’.
 Add 100 µl of serum to both tubes 1 and 2.
 Use a clean pipette to mix the 1/2 dilution of tube 2 several times and then transfer
100µl to tube 4. Tube 4 will attain a 1/4 dilution.
 Use a clean pipette to mix the 1/4 dilution several times and then transfer 100 µl to tube
8.
 Continue the process of all dilutions (512).Remove 100 µl from tube 10 (512) and
reserve in a clean tube if the titration needs to be continued.
 Add 50 µl of specific red cells suspension to each tube. Mix the tubes well.
 Incubate the tubes in 30 degree incubation.
 After incubation, resuspend the mixture from each tube and wash 3 times in saline.
 Add one drop of AHG to each tube. Mix well and centrifuge all tubes.
 Starting with the last tube, examine each tube macroscopically for agglutination. Grade
the positive tubes and record reaction.

NOTES AND INTERPRETATION:

 If testing a pregnant female, each month serum should be compared to the previous
month.
 Prozone phenomenon may occur so the first tubes may have a weaker reaction than the
more diluted serum. Read the most dilute tubes first and then shake out the other
tubes.
 Careful pipetting is essential.
 Cells with known antigens may have different reactivity and therefore the serum from
each month use the same cells.
INTERPRETATION:

 Observe the highest dilution that produces 1+ macroscopic agglutination.

 The titer is reported as the reciprocal of the dilution level: 32 not 1/32.
 A rise in titer would need to be at least 2 dilution increase between the current
specimen and the previous month.
BODY FLUID ANALYSIS:

(SEMEN ANALYSIS)
CLINICAL SIGNIFICANCE :-
A Semen analysis is performed in order to determine the fertility
potential of a male . At a minimum three basic parameters are assessed
sperm concentration , motility and morphology .

PROCEDURE :-
Semen analysis involves following steps .

 In the frist 5 mins: placing the specimen container on bench or

incubator (37 C) For liquefaction .
 Between 30 & 60 mins:
Assessing liquefaction and appearance of the semen .
 Measuring volume of semen
 Measuring semen Ph ( if required )
 Wet preparation for microscopy
 Assessing sperm count.
 With in 3 hours: Sending samples to microbiology lab
(if req.)
 Assessing smear for morphology.

SAMPLE COLLECTION:

 Sample should be collected after a minimum 2 days and maximum

7 days of sexual abstinence.
 Label the sample.
 Sample should be ejaculated into a clean wide mouthed
container.
 Place the container in an incubator (37C for liquefaction).
 Safe handling of specimens: