Line Immuno Assay

Th INNO-LIA HIV-1/11 score is a line Immuno Assay (LIA) to confirm the presence of
antibodies against the human immunodeficiency virus type 1 (HIV-1),including group 0 and
type 2 (HIV-2) in human serum or plasma .The score also differentiates between HIV -1 and
HIV -2 .The assay was performed on the essence that the specimens were found to be
reactive using an anti-HIV screening procedure .

Test Principle

Recombinant proteins and synthetic peptides from HIV-1 and HIV-2 and a synthetic peptide
from HIV-1 group O are coated as discrete lines on a nylon strip with plastic backing .Five
HIV-1 antigens are applied : sgp 120 and gp41 ,which detect specific Ab to HIV -1 and
p31 ,p24 and p17 ,which may also cross react with Ab to HIV-2 .

The antigens gp36 and sgp 105 are applied to detect antibodies specific to Hiv-2 .

Background Control
B+
1+ INNO -LIA HIV-1/11
+/-
Score test strip.
Sgp 120
Gp 41
P31
P24
P17
Sgp 105
Gp 36
3.Haemotology Section

Complete Blood Count Using Cowter Counter Act 5 diff

Principle : The cowter Act 5 diff analyzer uses AcV (Absorbance cytochemistry and volume )
technology which is used to analyze the final RBC /Plt dilution and the WBC /BASO
dilution. This electronic method of counting and sizing particles is based on the fact that cells
,Which are poor conductors of electricity,will interrupt a current flow .The impedance
variation generated by the passage of non-conductive cells through a small ,calibrated
aperture is used to determine the count (no of particles and size) of the particles passing
through the aperture within a given time period .

Complete blood count (CBC) is performed on patients to identify normal and abnormal bloos
parameter results ,which guide the physician/clinician on the appropriate course of
therapeutic management to patients and also to identify patient results that require additional
studies .For effective use of the ACT 5 diff the following consists of the reagents used :

i. Act 5 diff Diluent solution

ii. Act 5 diff Fix
iii. Act 5 diff WBC lyse
iv. Act 5 diff Hgb lyse rinse

Power Uping

Switch on the CPU

Switch on the monitor .Wait as Booting sequence follows

Press CTRL +ALT +DE;ETE to log on

Log on with the user name as BCI and password as 123

Switch on the analyzer and let the analyzer to power on

Press Enter or Ok and waitas the computer compacts database

Log in as operator with the operator’s initials .

Allow sufficient time for the computer to complete internal checks

After log in ensure the analyzer connection status icon is green.

Interpretation

WBC 5.4 Range 3.3/11.0

RBC 4.9 Range 4.10/6.10

HGB 13.2 Range 12.0/16.0

HCT 40.7 Range 37.0/48.0

MCV 83L Range 86/98

MCH 27.0 L Range 27.0 /32.0

MCHC 32.5 Range 31.0/35.0

RDW 14.4 Range 11.0/16.0

PLT 228 Range 130/550

MCV and MCH are abbreviated are abbreviated as L because they are Low from the range
of 86/98 and 27.0 /32.0 respectively . Initialization of the Beckman Cowlter counter takes
roughly 15 minutes.

4. HIV-R laboratory clinical chemistry section

i. COBAS Integra 400 plus

The COBAS integra 400 plus majorly works on these 4 principles :
i. Absorbance photometry
ii. Turbidimetry
iii. Fluorescence polarimetry
iv. Ion-Selective electrode measurements (ISE)
Absorbance photometer measures light intensity at different wavelengths .The light beam
from the absorbance halogen lamp passes through cuvette and then into a photodiode array
where the measurements are made .

Turbidimetry measures the concentration of an analyte based on the principle or turbidity

/cloudiness .

The Fluorescence photometer (FP) makes measurements based on the principle of

fluorescence polarization .

Controls used in the COBAS integra 400 plus include :

i. PCCC1-Preci Control Clin chem Multi-1

ii. PCCC2- Preci Control Clin chem Multi-2

ii).Serum Separation

Procedure :Centrifuge for 25 minutes at a speed of 1700 rpm .To calculate the number of
rpm necessary to attain 100og,used the formula:

9450
RPM= ,where r is the distance in centimeteres from the centre of rotation to the
√r
bottom of the test tube .

Principle :Blood natural clotting mechanism or silicon and clot activator allow blood to
clot and express serum.

The difference in the densities for serum ,separator gel and clot causes separation during
centrifugation.

iii) First Response HIV-1/2 card test .

This test is intended for the use by health care professionals and is
qualitative ,screening ,in vitro diagnostic test for detection of antibodies specific to HIV1
(including groupO) and HIV-2 in human serum ,plasma or venous and capillary blood .

Assay Principle

Is a lateral flow chromatographic immune assay .The test consists of :

  A purple colored conjugate pad containing HIV-1 and HIV-2 specific recombinant
antigens (gp41 including group O and p24 ) for the detection of antibodies to HIV-1
and HIV-2 specific recombinant antigens (gp41 including group O ,gp36 and (p24)
and control protein conjugated with colloidal gold particles
 A nitro cellular membrane strip containing two test lines (1 and 2)and a control line
(c)\
Test line 1 is precoated with HIV-1 recombinant antigens (gp41 including group O
and p24) for the detection of antibodies to HIV-1 .
Test line 2 is precoated with HIV-2 recombinant antigen (gp36) for the detection of
antibodies to HIV -2.
The control line is precoated with a control line protein.

2 C

When an adequate volume of test specimen is dispensed into the sample well of test
cassette ,the specimen migrates by capillary action across the strip .HIV-1 Abs ,if present in
the specimen ,migrate through the conjugate pad where they bind to the HIV-1 conjugates .

The immune complex is then captured on the membrane by the precoated HIV-1 antigen
forming a purple colored line at test line I indicating a HIV-1 antibody positive or reactive
test result .

HIV (Human Immunodeficiency Virus ) is recognized as the etiologic agent of AIDS .The
virus is transmitted by sexual contact ,exposure to infected blood ,certain bodily fluids or
tissues and from mother to foetus or child during the prenatal period .

The clinical diagnosis of HIV has been done by detection of HIV-1 and HIV-2 antibodies in
human plasma ,serum or venous /capillary whole blood by immunoassay .
Researchers have constructed HIV-1 and HIV-2 genes for the expression of recombinant
antigens in bacterium systems such as E.coli and focused on HIV-1 and HIV-2
proteins ,which are definitely immunogenic .The major immunoreactive antigens of these
proteins have been reported to have HIV-1 gp41 ,p24 and HIV-2 gp 36 based on Western
blot analysis .

First responsive HIV-1-2-0 Card test is a 3rd generation HIV immunoassay .The design of the
3rd generation assays allows the detection of HIV specific lg G as well a lgM ,which may
occur easily in infection.

Iv )Pregnancy Test

Pregnancy test check for a hormone called chlorionic gonadotropin (hCG)

Pregnancy test -HPTN 084 Samples

-Urine sample is collected from the patient

-Taken to the laboratory for testing

-Time set for 3 minutes

-Using a pipette ,draw urine from the bottle then added drops of urine on the pregnancy test
kit .

-Waited for another 3 minutes to observe results

C C

T T

Negative Positive
v)Urinalysis

By use of Combur 10 test

Procedure : In a sample of urine ,dip the COBAS strip into it .Then observe colour
changes on each bands on the test strip .Compare the strips with the Combur10 Test
strip card

Urinary cells ,Urinary casts ,urinary Crystals .

Urinary cells

Erythrocyte ,Dysmorphic ,Leukocyte ,Monocyte ,Renal tubular epithelial

cell,spermatozoa ,squamous epithelial cell and transitional epithelial cell.

Urinary casts

Fatty casts ,cellular cast ,granular cast, hyaline cast, erythrocyte, waxy cast.

Urinary crystals

A+ Acid pH

 Crystine Crystals
 Sulfonamide crystals
 Uric acid crystals
 Amorphous urate crystals

A+ neutral pH
 Bilirubin crystals
 Calcium Oxalate Crystals
 Cholesterol Crystals
 Lucine crystals

A+ alkaline pH
 Ammonium Biurate Crystals
 Ammonium Magnesium Phosphate Crystal s
 Amorphous Phosphate Crystals
 Amorphous phosphate crystals.
5. Cell Separation section
Plasma separation

Plasma is the cloak, yellowish fluid portion of blood, lymph or intramuscular fluid in
which cells are suspended. It differs from serum in that it contains fibrin and other
soluble clotting elements. Plasma also contains proteins and electrolytes including the
liquid blood plasma and interstitial fluid.
Procedure:
 Receive whole blood sample in EDTA tubes at room temperature
 Using a sample rack, carry the samples to the centrifuge.
 Balance the sample in the centrifuge
 Centrifuged the sampler of 2200 rpm for 10 minutes
 After centrifugation, the samples separate into a layers of plasma and RBC.
 Store plasma temporarily at -15oc to -30oc overnight or until they are shipped ,store
plasma samples in the -80oc freezer for long term storage.

Whole blood
Plasma

RBCs

Peripheral Blood Mononuclear cells (PBMC) separation. Principle: Freshly collected of cruo
preserved PBMCs, are used for the evaluation vaccine of anti-retroviral therapy-induced
cellular immune responses ,HIV-associated changes in immune response, and recovery of
replicated competent virus. There assays require PBMC that have been isolated and
cryopreserved under strictly defined conditions that ensure optimal recovery and
functionality.
One currently accepted technique for mononuclear cell separation as referred to Ficall
Hupaque Centrifugation Method, which employs a liquid density gradient medium of Ficoll
400 and sodium diatrizoate of sodium metrizoate solution (LSM).

The technique I used with anticoagulated blood collected by routine. Phlebotomy using the
advantage of the difference in density between mononuclear cells and other blood elements.

Ficoll - Hypaque density gradient facilitates the purification of PBMCs. During

centrifugation PBMC and platelets collect on top of the Ficoll Hypaque layer, while red cells
and granular lymphocytes with a higher density collect of the bottom.

Preparation of Dried Blood spot

Received whole blood sample in EDTA tubes.

Using Pasteur pipettes, measure 25 µl of whole blood

Drop the blood on Whatman card.

The card has 5 blood spots circular

Leave the card to day overnight or after 24hours

The card is reliable and used for the storage of blood for a longer period of time. This is
because it contains protein in it that preserve and retain the cell Components of the blood for
a longer period of time.
CONCLUSION

Throughout my stay at the Kenya Medical Research Institutes there has been ups and downs
throughout the process.. First of all I must give gratitude to all the personnel that aided my
training at the institute. The environment had been conducive and the tranquility that is
suitable for better learning and training. The institute has got also modern and advanced
equipment’s that makes it effective and efficient for carrying out experiments. The training
personnel, that is the staff has been so supportive and they have been willing at all cost to
give out a helping hand in training and instilling knowledge to our studies.

The only disadvantages has been observed during our field work when collecting mosquitoes,
they would sometimes bite us. That made me fear for possible infections such as malaria
"that be caused by the biting of mosquitoes. T

he training has been so successful since the first day and I have learnt a lot ,not only new
skills and knowledge ,has widened my understanding of some physiological functions

REFERENCES

Clinical Hematology Atlas, 3rd Edition by Jacqueline H. Carr and Bernadette F. Rodak 1999
Hematology Basic Principle and Practice by Ronald Hoffman,

Edward J. Benz, Leslie E. Silberstan, Helen Heslop, Jeffrey Wertz 6th Edition

Lehninger Principles of Biochemistry , Biochemistry, seventh edition by

David L Nelson and Michael M. Cox.

Malignant by Vinayak K Prasad MD, MAH 2020.