Routine investigations in AIHA include reticulocyte count, serum LDH, and serum haptoglobin. An increased reticulocyte count, elevated serum LDH, and decreased serum haptoglobin indicate hemolysis. Reticulocyte count measures immature red blood cells in the blood to assess bone marrow response. Serum LDH is released during red blood cell breakdown and elevated levels suggest hemolytic anemia. Serum haptoglobin binds free hemoglobin but is consumed in hemolytic diseases, so low levels indicate intravascular hemolysis. Together these tests help evaluate the severity and stage of hemolytic anemia.

1. Routine investigations in AIHA:
Reticulocyte count, sr.LDH,
sr.Haptoglobin
Dr Deepa Nagarajan
Clinical associate (Pathology)
LHRC

2. Reticulocyte
• Reticulocytes are young or juvenile red cells
released from the bone marrow into the
bloodstream and that contain remnants of
ribonucleic acid (RNA) and ribosomes but no
nucleus.
Stages of Erythropoesis
Dr Deepa Nagarajan

3. Gilmer and Koepke defined reticulocyte in 1976 as
Dr Deepa Nagarajan

4. Stages of maturation of
reticulocytes (Heilmeyer
and Westhaeuser, 1932)
• Stage I: Dense cohesive reticulum in
non-nucleated red cell (0.1% of
reticulocyte count in normal individuals)
• Stage II: Extensive network of loose
reticulum (0.7% of reticulocyte count in
normal individuals)
• Stage III: Small reticulum along with
scattered granules (32% of reticulocyte
count in normal individuals)
• Stage IV: Scattered granules (61% of
reticulocyte count in normal individuals).
Dr Deepa Nagarajan

5. Dr Deepa Nagarajan

6. Methods :
A. Manual
B. Automated
Manual
• A. Supravital staining method, B. Fluorescent method, C.
Miller ocular method
Supravital staining method :
It refers to staining of cells in a living state i.e blood is mixed
with the stain and stain enters the cell in living condition,
before they are killed by fixation or drying or d/t passage of
time.
Reticulocyte contains ribosome and RNA ,which get stained
with supravital stain and appear blue filamentous or granular.
Reticulocyte count is then performed by manual method.
I) Brilliant cresyl blue- An oxazine dye
II) New Methylene blue- An thiazine dye
III) Azure B
Dr Deepa Nagarajan

7. Principle
• SPECIMEN: EDTA –anticoagulated blood .
• PROCEDURE: 1) Take 2-3 drops of dye solution
into a small test tube. 2) Add 2-3 drops of
patients EDTA-blood and mix. 3) Incubate at 37⁰C
in a waterbath for 15-20 minutes . 4) Mix and
prepare smear. 5) Air dry and observe under
microscope.
• By using oil immersion objective choose an area
of film where the cells are undistorted & staining
is good.
Dr Deepa Nagarajan

8. CALCULATIONS:
Count at least 1000 RBC’s including reticulocytes
which are easily identified with blue granular or
reticular precipitate in the cytoplasm.
Reticulocyte count is express as % of the red cells.
Retic count% = Total no. of Reticulocytes/Total no.
of RBC’s X 100
Normal range: 0.2-2%
Dr Deepa Nagarajan

9. Reticulocyte
associated
parameters:
Absolute Retic count= Retic
count% X Red cells
• Reticulocyte index is necessary for correction for anemia.
Dr Deepa Nagarajan

10. Other red cell inclusions can be seen in the
brilliant cresyl blue/new methylene blue
smears:
a) HbH bodies:
• Round inclusion bodies which stain
greenish-blue( golf ball inclusion).
• Globin ch ppt
• They are found in alpha
thalassaemia or Haemoglobin H
disease.
b) Heinz bodies:
• Seen as blue granules ,variable in
size, lying to one side of the cell
near the membrane.
• Denatured hb.
• They are found in G6PD deficiency
Dr Deepa Nagarajan

11. Automated
method:
Recent methods
based on flow
cytometry have
been introduced
which are rapid
and more precise
• Various dyes and fluorochromes combine
with the RNA of reticulocytes.
• Following binding of the dye, fluorescent
cells can be enumerated using a flow
cytometer.
REF: SYSME_15619_SEED_Haematology_EN (sysmex-europe.com)
Dr Deepa Nagarajan

12. • The dyes used in the different systems include auramine O (Sysmex),
tliiazole orange (ABX), CD4K 530 (Abbott), as well as nonfluorescent
dyessuch as oxazixie 750 (Bayer-Technieon) and the traditional New
methylene blue (Recbman-Cosilter, Abbott)
• The fluorescence flow cytometry method allows the classification of
reticulocytes into three maturation stage, which are defined by the
RNA content of the reticulocytes. On the analyser; giving three
different fluorescence intensity: LFRS, MFR and HFR
Dr Deepa Nagarajan

13. Dr Deepa Nagarajan

14. Retic channel :
Three Reticulocyte
parameters
• Reticulocyte count (RET #, %)
indicates the quantity of
circulating reticulocytes
• Immature Reticulocyte
Fraction (IRF) indicates the
rate of production of
reticulocytes
• RET-He indicates cell
hemoglobinization, reflecting
quality of the newly produced
reticulocytes.
REF: SYSME_15619_SEED_Haematology_EN (sysmex-europe.com)
Dr Deepa Nagarajan

15. Immature Reticulocyte fractions-
• IRF is the sum of MFR and HFR i.e the immature reticulocytes and is referred to
as the ‘reticulocyte maturation index’.
• Is an early and sensitive index of erythropoiesis.
• The IRF percentage increases after only few hours, the reticulocyte count
increases after two to three days.
• If the IRF value does not increase during the treatment of deficiency anaemias, it
indicates a lack of response to therapy.
• During reticulocytopenia, it facilitates early identification of marrow regeneration
in patients undergoing bone marrow transplantation or chemotherapy.
• Reference range IRF: 3.1 – 13.4 %
Dr Deepa Nagarajan

16. REF: SYSME_15619_SEED_Haematology_EN (sysmex-europe.com)
Dr Deepa Nagarajan

17. RET-He
gives the Hb content of the developing reticulocytes, young RBCs and
thus offers real-time information on iron supply to erythropoiesis.
Provides information on the current bioavailability of iron.
RET-He is not affected by the "acute phase reaction’.
It is useful for early detection of iron deficiency.
It is also used for monitoring of erythropoietin and iron therapy
Reference range: 30.3-36 pg
Dr Deepa Nagarajan

18. 1
4
4
2
3
1
Ret% :21.39%↑
IRF: 52.3%↑
RET-He:33.4 n
DCT: ++
Sr LDH: 783 U/ L ↑
Sr haptoglobin: <10 ↓↓
LHRC case study
Dr Deepa Nagarajan

19. Causes of increased reticulocyte count
(reticulocytosis):
• Hemolytic anemias
• Blood loss
• Following specific therapy of nutritional anemia (like iron in iron deficiency anemia,
folate in folate deficiency anemia, Vit B12 in B12 deficiency anemia)
• Hemoglobinopathies, e.g. sickle cell anemia
Dr Deepa Nagarajan

20. Causes of decreased
reticulocyte count
(reticulocytopenia):
Aplastic anemia and pure red cell aplasia
Bone marrow infiltration (leukemia, lymphoma, myelofibrosis,
metastatic malignancy)
Renal disease
Anemia of chronic disease
Alcoholism
Myxedema
Ineffective erythropoiesis: Megaloblastic anemia, sideroblastic
anemia, thalassemia, myelodysplasia
Following blood transfusion
Dr Deepa Nagarajan

21. Serum Lactate
Dehydrogenase(LDH)
enzyme
• Is an integral part of the glycolytic pathway of
carbohydrate metabolism
• LDH is present in virtually all body tissue
paticularly in heart, liver, muscles, kidneys and
RBCs.
• The LDH in serum can be separated into five
isoenzymes based on their electrophoretic
activity.
• Each isoenzyme is a tetramer composed of
two different subunits.
• These two subunits have been designated
heart and muscle, based on their polypeptide
chains.
• There are two homotetramers LDH-1 (heart)
and LDH-5 (muscle), and three hybrid
isoenzymes.
Dr Deepa Nagarajan

22. PRINCIPLE
UV assay:
Lactate dehydrogenase catalyzes the conversion of L-
lactate to pyruvate with NAD being reduced to NADH in the
process.
L-Lactate +NAD+→ Pyruvate + NADH + H+
The rate of NADH formation is directly proportional to
catalytic LDH activity. It is determined by measuring the
increase in absorbance at 340nm.
Reference range: 135- 225 U/L
Dr Deepa Nagarajan

23. Elevated levels:
Serum levels Associations
Mild MI, Leukemia,hemolytic anemia & non viral
hepatitis
Moderate Muscular disorders, nephrotic syndrome, &
cirrhosis
High Megaloblastic anemia, disseminated ca, shock
Ref :LD - Clinical: Lactate Dehydrogenase (LDH), Serum (mayocliniclabs.com)
Dr Deepa Nagarajan

24. Serum
Haptoglobin
Haptoglobin is a transport and acute phase protein which is
synthesized in hepatocytes.
ls a glycoprotein which consists of two alphalight chains and two
betaheavy chains.
The genetic polymorphism of the alpha chains leads to three
phenotypes Hp 1-1, Hp 2-1 and Hp 2-2 differing in molecular weight.
Various methods including nephelometry, radial immunodiffusion
(RID) and turbidimetric methods are available for the determination
of haptoglobin.
Reference range: 30-200 mg/dL
Dr Deepa Nagarajan

25. Dr Deepa Nagarajan

26. • Haptoglobin binds hemoglobin in a strong haptoglobin-hemoglobln complex (Hp-Hb), the
hemoglobin resulting from pathologically elevated hemolysis.
• These complexes are deposited in the hepatocytes, the deposition process having a half-
life of less than 10 minutes.
• Hemoglobin is enzymatically metabolized and haploglobin is liberated after
approximately 3 days.
• Complex formation and the extremely rapid elimination from circulating blood prevent
the ocurrence of hemoglobinuria with excess renal loss Iron.
• A reduction in the level of free haploglobin is indicative of intravascuar hemolysis
Dr Deepa Nagarajan

27. Indications
Assessment of the severity and stage of Intravascular hemolysis
Evaluation of acute inflammatory processes
Test principle
• Immunoturbidimetric assay.
• Human haptoglobin forms a precipitate with a specific antiserum
(immunological agglutination) which is determined turbidmetrically.
Dr Deepa Nagarajan

28. Clinical significance:
Increases in response to stress, infection, acute inflammation and tissue
necrosis.
After hemolytic episode , sr haptoglobin concentration falls
Low levels may be seen in severe liver diseases.
Also useful for serially monitoring patients who have a slow but steady rate of
RBC breakdown such as mechanical heart valves and strenuous exercise
Ref: HAPT - Clinical: Haptoglobin, Serum (mayocliniclabs.com)
Dr Deepa Nagarajan

29. References
• Practical Haematology-Sir John V Dacie & SM Lewis.
• Essentials of Haematology-Shirish M.Kawthalwar..
• Atlas and test of hematology 4e Dr.Tejinder Singh
• Reticulocyte count (slideshare.net)
• RET-He_A_Parameter_with_Well-Established_Clinical_Value.pdf (sysmex.com)
• SYSME_15619_SEED_Haematology_EN (sysmex-europe.com)
• Microsoft PowerPoint - 2015 Geiger Sysmex Ret He V5 Mar 3 2015-FINAL
EDITS.ppt [Compatibility Mode]
• Henrys Clinical Diagnosis and Management by Laboratory Methods 23th Edition
• mayocliniclabs.com

30. Thanks