2. 1.CBC – Few practical points
■
■
■
Commonest lab test in clinical practice
Results are not specific to a particular disease
Requires interpretation based on patients clinical condition
■
■
Peripheral smear -most important but negleted part of CBC
All three cell lines and subtypes of WBC need proper
evaluation
1. WBC count and DC
2. RBC count ,Hb% and Indices
3. Platelet count and Indices
5. Increased WBC
Acute stress of various causes
■ Infection, tissue necrosis
■ bone marrow malignancies,
inflammation
Decreased WBC
■ Infections
■ conditions or medications that
suppress or weaken the immune
system or bone marrow
6. Neutrophils -60%
■First line of defense against infection
Two types:
■ Bands (0-3%) - immature
■ Segs (31% - 57%) – mature
■ Neutrophilic leucocytosis with >20% band-
Acute bacterial infection –(Not a rule)
Neutrophilia indicates stress of
■ Acute infection-Bacterial/viral
■ Inflammation
■Others – Burns ,acute asthma
Neutropaenia indicates marrow suppression
■ Typhoid,Viral infections
7. Absolute Neutrophil Count (ANC)
■ The real number of white blood cells (WBCs)
that are neutrophils.
■ Anormal ANC is about 3,000-5,000.
■ <1,000 greater risk for infection.
10. Leukocyte abnormalities –limitations…
■ TC -low predictive value due to wide range of
normal count (8000 to 20,000/cu.mm).
■ Non-specific-infective /non-infective inflammation
■ Non specific-bacterial /viral.
■ Leukamoid reaction (WBC count > 50,000) should
not be mistaken for malignancy like leukemia.
(Leukocyte alkaline phosphatase increased in LR)
13. Neglected part – MPV & RDW
MPV reflects the average size of platelets
Predicts functionally good platelets
■ High MPV in a person with a low platelet count
suggests the bone marrow is producing platelets
and releasing them into circulation rapidly.
■ Low MPV with low platelet counts - due to a
disorder affecting production of platelet by the
bone marrow
PDW reflects how uniform the platelets are in size.
■ Normal PDW - platelets are mostly same size
■ High PDW - platelet size varies in size
14. N +++ +++ N ++ Sys.Inflammatory dis
N ++ ++ 0 N/L Acute viral inf
N L ++ o L Typhoid
N +/- ++ N N/L Chronic infections
Low +/- ++ N N/L Malaria
Low +++ ++ N Low AL L
+
High +/- ++ 0 Low Dengue
CBC in common clinical conditions
Hb WBC P L E Plt Disease
N +++ +++ 0 N Acute bacterial inf
15. Alerts
■ High Hb +Pcv(>20%) –Dengue fever
■ Low WBC + High Hb - Dengue
■ High WBC + High lymphocytes – ALL
■ Low WBC + Low platelets + low Hb – Bone
marrow disease
■ Low lymphocytes <2700 -Immunodeficiency
16. CBC in fever-limitations
■ CBC done very early during a febrile illness does not
help much
■
■
■
Interpret only in conjunction with the clinical picture
It should be repeated if fever persists without diagnosis
Serial counts (eg) Dengue
17. Case 1
■ 5 yr old boy –Acute onset of high grade fever-2days
■ Interfebrile state –Sick
■ Poor response to supportive treatment
■ D2 –CBC-Hb 11.5g%HCT-34 TC-7600 DC- P62 L36 E 0
■ Plt-1,60,000 , Urine R/E –Normal
■ D3-C/O Abd.pain,headache,2 episodes of vomiting
■ Sick looking,Temp – 104 F ,Moderate dehydration
■ CRFT-3sec ,Low pulse volume
■ Flushing-white island in the red sea
■ Admitted –Supportive treatment –Rpt CBC -Hb-13.4g%
HCT 41.4 TC 3100 DC P38 L 61E0 Plt- 95,000
18. ■ Prov
vi
is
si
io
onal diagnosis
?Dengue fever
■Treatment - WHO guidelines/frequent assessment
Logical Lab investigations in Suspected dengue
1.For assessment and management
■ Serial –Hb%, Hct, Platelet count
■ Na,BUN,LFT
■ DIC Panel
■X-ray chest, USG abdomen
2.For confirmation of diagnosis
■ Serological tests
19. CBC in dengue
■ Serial Hb%, Hct, Platelets – Atleast every 24 h
■ Q 4 h in severe cases of dengue.
■ Rise in Hct level > 20% from baseline is a sign of
haemoconcentration - Shock
■ Rapid decrease in platelet count +rising Hct =
progress to the plasma leakage/critical phase
■Leukopenia(<5000)+Positive tourniquet test in
Dengue endemic area-Positive pred.value-70-80%
20. Confirmatory tests
■ Diagnosis is clinical …
■ Monitoring with simple tests
Confirmatory tests ??
■ To R/O dengue like illness
■ Primary /Secondary infection
21. Lab investigations for confirmation of diagnosis
■ Isolation of virus
■ RT-PCR - considered as "gold-standard“
■ Serological tests -1)NS1 2)IgM/IgG
■NS1 Antigen
1)Microwell ELISA TEST
2)Lateral Flow Rapid Test (LFRT)
■ IgM/IgG
1) E/M-specific capture IgM and IgG ELISA
2) Rapid chromatographic test -IgM & IgG
22. ■ NS1 antigen –Non structural protein
■ High concentration in the acute-phase of primary and
secondary dengue virus infections
■ Up to 9 days after the onset of illness
NS1 test ELISA RAPID TEST
Time required 2 hr 15-30 min
Cost/availability Costly / + Cheap / -
Specificity 100% 100%
Sensitivity 82% 72%
Primary/secondary - -
26. Neonatal septic screening
Components
1. Total leukocyte count (<5000)
2. Absolute Neutrophil count
3. Immature to mature ratio (I:T Ratio) >0.2
■
■
4. CRP
5. Micro ESR
Septic screen is considered positive if 2 or more
parameters are abnormal (Sensitivity 93%
Specificity 83%)
Septic screen does not include blood c/s
,CXRay,or lumbar puncture –These are definitive
tests
27. Sepsis screening – limitations
■ Interpret sepsis screen taking into consideration
the risk factors/gestation of infant/clinical
status/timing of screen
■ Taken isolation none of the tests are definite
indicators of sepsis
■ Do not use septic screen as a substitute for
blood culture
■ Do not use SC to decide duration of antibiotic.
28. C Reactive Protein
■ Produced by hepatocytes
■ Healthy individuals <1mg dL
■ CRP starts to rise within 12 to 24 hours of onset
of sepsis (Earlier than the other acute phase reactants.)
■ Can rise 1000 fold within 24 hrs
■ Half life<24hr
29. CRP Practical points…
■
■
Best studied in neonatal sepsis
Serial CRP levels are useful to exclude diagnosis of
neonatal sepsis. If two CRP measurements 24 h apart
are <10 mg/L.-Negative predictive value >98%
■
■
■
■
Positive response needs correlation with other
parameters -Positive predictive value <50%
It is not justified to start the antibiotics only on the ground
of positive CRP.
Serial measurement provide additional information on
the adequacy of treatment
Trend of CRP is more important than a single CRP value
in monitoring the activity of inflammation.
30. Micro-ESR
■ Simple marker for neonatal infection.
■ Not a very reliable marker.
■ Its normal value is 6 mm -first 3 days of life.
■ End of first month, upto 11 mm.
■ >15 mm is suggestive of infection.
32. Case 3
■ 7yr old boy – fever 6days
■ Continuous - high grade - rising trend
■ D1-3 : No other localizing symptoms/signs
■ D 4-5 –Vague abd pain ,loose stools
■ D6 -Toxic ,104 F, Tongue - coated
■ P/A-Liver 2cms /span 8cms,Spleen 1cm /
soft
■ Provisional diagnosis
■ Enteric fever
33. ■ CBC,Urine R/E, Blood c/s
■ Urine R/E –Normal
■ CBC-TC-3400/cmm.
■ DC-P 60 L 38 E 0 M 2
■ Hb 11.6 HCT 33.8
■ Platelets 142000
■ Clinical +low leukocyte count with
eosinopenia points to possible
Enteric fever
34. ■ Blood culture -gold standard
■ Sensitivity –highest in the first week of the
illness
■ Reduces with advancing illness.
■ Overall sensitivity - 50 %
■ Drops with prior antibiotic therapy
■ Bone marrow culture is a highly sensitive
even in late stages of the illness and with
prior antibiotic therapy.
35. Blood c/s in practice
■ Gold standard for bacteremias
Indications
■
■
■
Age <3mon with suspected infection
Suspected Enteric fever
FUO
■
■
Febrile nutropenia/ Immuno compromised child
Hospital acquired infection
Practical points
■
■
■
■
■
Cleaning -70% Alcohol / 2% chlorhexidine
Universal precautions
Amount of blood – 1-5ml
Blood :Media – 1:5
Preferably before antibiotics
36. Blood culture types
■ Conventional
Limitations
1. Labor-intensive
2. Time consuming
3. Sub culture - each sample manually after 12,24,48 hr
■ Automated blood culture systems
1. Bacterial growth is detected by microbial production
of CO2
2. BACTEC / BacT-alert / MGIT / BACTEC +
37. Automated culture
Advantages
■ Improved recovery of organism
■ Rapid isolation
■ Better identification
■ Accurate susceptibility
Limitation
■ Non availability
■ Cost
BETTER
MANAGEMENT
39. Widal test
■ Most widely used serological test
■ Order this test only after 5-7 days of fever
■ Tube method is better than slide method
■ Both H and O antibodies of 1in 160 dilution
should be taken as cut off value of diagnosis.
■ H antibodies once positive can remain positive
for long time.
40. Serum Widal –Limitations….
■ Timeline - Send only after first week of fever
■ Poor sensitivity- false negative 30%
■ Previous antibiotic treatment
■ Poor specificity - endemicity & anamnestic reasons
■ Method : slide test – Not reliable
■ Vaccination
whole cell vaccine-High baseline anti-o &anti-H
(Vi-polysaccharide vaccine does not interfere )
■ Inter laboratory variations
41. Typhidot test
■ A dot enzyme immunoassay that detects specific
IgM and IgG antibodies - 50KD outer membrane
protein antigen of S. typhi.
■ Limitation: False positive results in endemic areas.
-Persistence of high IgG levels
■ Typhidot-M® - only IgM is detected. IgG is
inactivated
■ Typhidot-M® -sensitivity (>93%)
42. Molecularmethods
PCR for Typhoid
■ Superior sensitivity than culture
■The turnaround time < 24 hours
Limitations
■ Clinical utility - inadequately evaluated
■ False positive –Vaccination , Old infections
■ Costly
43. Tubex test:
■ Tubex test is simple one-step test taking 2minutes
■ It detects only IgM and not IgG
■ Positive result -Suggests recent Salmonella
infection
IgMdipstick test
■ Based on the binding of S. typhi-specific IgM
antibodies to S. typhi lipopolysaccharide (LPS)
antigen.
■ It can be done in serum or whole blood and
requires incubation for 3 hours
44. Case-4
■ 4yr old girl -H/O High grade fever 3 days
■ Sick looking. No focus of infection
■ Poor response to symptomatic treatment
■ Admitted for evaluation
■ Supportive treatment
■ Investigations ?
■ CBC, Urine R/E
45. Case4–Labreport
CBC
■ TC-23,000/cmm
■ Hb 11 gm %
DC :P 82 L 19 E 0 M 1
HCT 33.7
■Platelets 3,31,000
Urine analysis
Nitrite +
■ Protein +
■ Leucocyte esterase +
■ Microscpy 40-50 / hpf
46. Diagnosis: ?UTI
■ Mild proteinuria
■ Significant pyuria >10 leukocytes per mm in a
fresh uncentrifuged sample, or >5 leukocytes
■
per high power field in a centrifuged sample.
Bacteria on Gram staining
47. Dipstick
■ Leucocyte esterase test –
Pyuria
■ Nitrite test - significant
Bacteriuria
■ Leucocyte esterase + Nitrite
- more sensitive
48. Clinicalapplication…
■ Useful in screening for UTI.
■ Combination of these tests has moderate sensitivity and
specificity for detecting UTI
■ Positive nitrite + leucocyte esterase
( sensitivity 72% and specificity 96%)
■ Positive Gram stain
(sensitivity 93% and specificity 95%)
51. Radio imaging studies
• Recommended for all children with UTI.
•Aim - to identify patients at-risk of renal damage, mainly those below 5 years of
age, with VUR or urinary tract obstruction.
52. USG inUTI
■ USG to be done in all cases of UTI
To L/F
1. Posterior urethral valve
2. Post void residual urine
3. Bladder hypertrophy
4. Hydronephrosis
5. Pelvicalyceal anomalies
53. MCU Scan
■ It will show anatomy of the bladder and the
ureters.
■ It is basically for bladder and VUR issues
54. A. The MCU showing a
dilated posterior
urethra, mildly irregular
appearance of the
edge of the bladder and
bilateral vesicoureteric
reflux into dilated
.tortuous ureters
B. Hydronephrosis
with 'clubbing' of
the calyces.
55. DMSAScan
■ Gold standard test for detecting renal cortical
scars
■ Can be done during acute phase of UTI
56. Case 5:Feverwithrash
■ 3 yr old girl with fever – 4 days ,
■ Skin rash – 2 days
■ Fever-moderate-high
■ Rash increased over 2 days and lead to black
patches over one ear lobe and one toe
O/E
■ Sick child with high fever
■ Maculonodular rash
■ Gangrenous patches-Ear lobe, one toe
57. Clinicaldiagnosis…
■ Rickettsial disease
■ Investigation
■ No rapid laboratory tests are available to
diagnose rickettsial diseases early in the
course of illness.
■ Gold standard –(IFA )Immuno fluorescence assay
■ Weile Felix - Useful and cheapest available tool for
the laboratory diagnosis of Rickettsial diseases in
India
■ ELISA / RT-PCR / dot blot immunoassay
58. ■ Weil-Felix is a nonspecific agglutination test
■ It detects anti-rickettsial antibodies in patient’s
serum.
■ If the titer is greater than or equal to 1:320 or
4-fold rise from baseline - POSITIVE
■ sensitivity(33%) and specificity(46%)
59. IFA -Immunofluorescence assay
■ Gold standard for diagnosis
■ IFA can detect IgG or IgM antibodies.
■ Blood samples taken early (7-10days) and late
(14-21days) in the disease are the preferred
specimens for evaluation.
■ Rising titres of IgM is indicative of a current
infection
60. Case6
■ 5yr old boy – Frequent cough cold & fever – 1yr
■ 6 episodes /yr
■ Each episode -2-4days of fever Cough&cold-5-10 d
■ No hospitalizations/No family h/o Atopy
■ Clinical exam - Normal
■ Inv-Hb 11g% TC 14300 P35 L62 E3 ESR 42 mm
■ Mx test- 10 mm Positive.
■ Rx –ATT – Referred for 2n
dO
p
in
io
n
■ No h/o contact , X – Ray chest –Normal
Diagnosis
■ Recurrent viral respiratory infection
61. Tuberculosis diagnosis
■ Golden triad
(Clinical features +Abnormal chest X-ray +Mx)
+ contact history/close exposure with an adult having
active TB in the last 2 years
■ Demonstration of AFB - Confirmatory diagnosis
AFB in smear/culture
■ Isolation of MTB –Clinical specimen/histopathology
62. Mantoux test
■
■
ID - PPD-2TU- 2-4 inch below
elbow
Transverse diameter of the
induration (not erythema)
■
■
measured- 48 -72 hours in mm
Positive >10mm-Infection
Reaction could be due to
1. Infection with tubercle bacilli
2. Cross sensitivity to
environmental mycobacteria
3. BCG-induced sensitivity
65. Automated liquid culture
BACTEC TB 460
■ Sensitive, specific and rapid culture method
■ Respiratory /non-respiratory specimens.
■ 200 viable M. tuberculosis bacilli could be detected in
12-13days
Mycobacterial growth indicator tube(MGIT) 960 TB:
■ Fluorescent technology - based on oxygen quenching
with a fluorescent dye.
■ Result - 7-10 days.
66. Direct molecular methods
■ Do not require growth of the bacteria,
■ Possible to detect M.tuberculosiscomplex within
3-5 hours.
■ Nucleic Acid Amplification assay tests
1. The Gen-Probe AMPLIFIEDTM
Mycobacterium tuberculosis Direct (MTD) Test.
2. The Roche AMPLICOR® Mycobacterium
tuberculosis (MTB) Test.
67. Limitation
■ Results of NAA tests are preliminary; the
mycobacterial culture is needed for species
identification, confirmation and for drug-
susceptibility testing.
■ Apositive result may be misleading as the NAA
test can amplify DNA from both viable and
non-viable organisms
68. Case 7
■ 5 yr old boy with fever 5days
■ High grade, intermittent, Chills & rigors +
■ Headache ,Vomiting +
■ O/E Temp 103 F
■ P/A Spleen measuring about 3 cm below costal margin
– firm , non-tender
■ No hepatomegaly
Diagnosis
Malaria
71. Blood smear for malarial parasite
Thickbloodsmearsaremostusefulfor
detectingthepresenceof parasites,
becausetheyexaminealargersampleof
b
l
o
■
o
d
.
Thinbloodsmearshelpsdoctors
discoverw
hatspeciesof malariais
causingtheinfection.
72. Disadvantages of Microscopr
■ Time consuming >60min
■ Labour intensive
■ Skilled lab technician
■ It cannot detect parasites sequestered deep in
the vascular compartment
73. QBC for MP
■ The blood is taken in a QBC
capillary tube which is coated with
acridine orange (a fluorescent dye)
and centrifuged
■ The fluorescing parasites can then
be observed under ultraviolet light
at the interface between red blood
cells and buffy coat.
■ This test is more sensitive than the
conventional thick smear and
■ > 90% of cases the species of
parasite can also be identified.
74. Rapid tests for malaria
■ Immunochromatographic tests based on the
‘Dipstick’format
■Detects plasmodium specific antigens in blood
sample.
■ Common RDTs available Parasight ,OptiMAL.
75. Targetedantigens
■ Histidine rich protein II of P. Falciparum (pfHRP-II)
■ Plasmodium aldolase- produced by all plasmodium
species.
■ Plasmodium lactate dehydrogenase(pLDH), is
produced by all four species of plasmodia.
■ Antibodies produced against pLDH either specific for
P.falciparum or P.vivax alone or a pan specific antibody
which reacts with all the four species of plasmodium.
Commercially available kit can detect falciparum and
vivax but cannot differentiate ovale and malariae.
76. Advantages
■ Rapid tests are easy to use with minimal training
■ Results are available within minutes.
■They can diagnose falciparum infection even
when the parasite is deeply sequestered.
77. Disadvantages
■ More expensive than microscopy
■ Limitations in species identification.
■ Persistent positivity even after effective
treatment. HRPII remains positive for 4 weeks
and pLDH remains positive for1 week.
■ No prognostic value- they can not quantify
parasites.
