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Year : 2019 | Volume : 7 | Issue : 3 | Page : 81 - 84  


Original Articles
Role of peripheral blood smear examination and manual platelet counts as an adjunct to automated platelet counts

Mohammed Moid Afzal 1, V Indira 2*, Colonel Ramanan Duraiswami 3 

1Associate Professor, 2Professor & Head, 3Professor, Department of Pathology Malla Reddy Institute of Medical Sciences, Suraram Cross Roads, Quthbullapur Municipality, Hyderabad 

Corresponding Author:

Email: indira_path@yahoo.com                                                                                                    

Abstract:

Background:  Assessment of platelet counts are an important part of the routine assessment of patients, especially in bleeding disorders, thrombocytopenia, and infections such as Dengue fever, which are associated with extremely low platelet counts. The platelet counts by automated Hematology counters are generally accurate, but at times, the machine cannot differentiate between platelets and non – platelet particles such as fragments of WBC, RBC and cellular debris.

Objective: We estimated the accurate platelet counts obtained by automated machine using impedance technology, after verification with platelet counts obtained by assessment of peripheral blood smears and manual counts using Neubauer chamber.

Methods: Blood samples of 236 patients reported to Malla Reddy Hospital Suraram Hyderabad, from 27 May 2019 to 09 June 2019 were studied. Patients were in the age of 4 days to 85 years, of both genders and had platelet counts below 1.5 x 103 / µl, estimated by Sysmex XNL 350. The counts thus obtained were verified by evaluation of Giemsa stained peripheral blood smears and manual platelet counts using improved Neubauer  chamber.

Results: The study revealed discordance between the machines generated platelet counts and manually evaluated platelet counts in 71 samples. In 63 samples the counts by Sysmex machine were lower than actual counts obtained by manual evaluation by a factor of 4 – 106 x 103/ µl. In only 8 samples, the machine generated count was higher than the manual count by a similar factor.

Conclusion: It is mandatory to verify abnormal platelet counts obtained by automated hematology analyzers, by examination of peripheral blood smears and manual counts using improved Neubergers chamber.

Key words: Accuracy of Platelet Counts, Pseudo thrombocytopenia, Platelet Satellitism, Myloproliferative Neoplasm, Giant Platelets

INTRODUCTION: 

Platelets are small (1-3 µ) discoid non nucleated cells containing red-purple granules produced in bone marrow by fragmentation of megakaryocytes; each produces 1000-3000 platelets. 1 Normal count of platelets is 1.5 lakhs-4 lakhs/µl with a life span of 7-12 days. 70% of the platelets are in blood circulation and 30% are sequestrated in the spleen and destroyed by macrophages in spleen. The main functions of spleen are hemostasis, thrombosis and they also play a key role in many physiological processes like wound repair and immune response. 2

The platelets in peripheral blood are heterogeneous with respect to size, density& staining characters, their morphology varies depending upon anticoagulant used and the method by which they are examined. 3 It is very important to evaluate accurate platelet count for management of thrombocytopenia. Mostly the automated counters provide accurate platelet counts but they are often unable to discriminate platelets from non platelet particles like fragments of WBCs, RBCs ,cellular debris, lipids, protein aggregates, platelet abnormal distribution, large platelets, small platelets, platelets clumps & very low platelet count< 30 x 109/l . 4, 5

The clinician has to correlate these platelet counts with the patients clinical presentation like minor bleeding, petechiae, epistaxis, sub conjunctival hemorrhage, hematuria, gross bleeding, and major bleeding like hematoma, vaginal bleeding, and gastrointestinal bleeding.

Hence it is mandatory to verify the result of automated counter platelet counts by peripheral smear examination and manual platelet count, especially when platelet counts are < 30 x 109/L or neonatal counts ≤ 100 x 109/L and first platelet count is ≤ 50 x 109/L by automated counter, it should be replaced by manual count method. 6. 7.

The following conditions cause thrombocytopenia, pseudo thrombocytopenia & thrombocytosis:

Thrombocytopenia:-Pregnancy, Heparin induced, immune thrombocytopenia, Idiopathic thrombocytopenic purpura, Infections: Dengue fever, EBV, HIV, Malaria. Neoplastic conditions like acute leukemia, Drug induced, Aplastic Anemia, Vitamin B12 and Folic acid deficiency, Exposure to Chemotherapy, Radiation, Hyper spleenism, disseminated intravascular coagulation.

Giant Platelets: - Myeloproliferative neoplasm (up to RBC size), Immune Thrombocytopenic purpura, Hyposplenism.

Inherited Disorder: - Bernard Soulier Syndrome, Grey platelet syndrome, May Hegglin Anomaly. 8

Pseudo thrombocytopenia: Anticoagulant EDTA causing platelets clumping, 9 sometimes platelets adherent to neutrophils due to anti platelet antibodies, Platelet ingested by neutrophils, Platelet Satellitism. 10

The presence of large platelets beyond upper limit may lead to under estimation of platelet counts. 11

Thrombocytosis: Myeloproliferative Neoplasms, Acute inflammatory stress, Chronic Bleeding, Spleenectomy

Hanseler et al using the H counter claimed that for platelet count less than 30,000/µL the automated chamber should be replaced by manual counting chamber procedure.

METHODS:

We compared the platelet counts obtained by fully automated analyzer Sysmex XNL 350 using impedance technology with platelet counts obtained by peripheral blood smear and manual counting by improved Neubauer counting chamber and assessed in 1650 cases and found low platelet counts (<1.5 lakh) in 236 cases admitted to or attending outpatient department of Malla Reddy Hospital Suraram, Hyderabad from 27-5-2019 to 9-6-2019 with age group of 4 days to 85 years.

Initial platelet count done using blood sample obtained from patients collected in Sodium (EDTA) as per the standard operating procedure. In 236 cases with low platelet count peripheral blood smear by Giemsa stain prepared &examined under light Micro scope (*100x) & Manual count of platelet counts done as per standard protocol using improved Neubauer counting chamber.

The results analyzed to obtain the degree of concordance between manual& automated plate counts.

METHODS:-

  1. Peripheral blood smears stained by Giemsa stained are thoroughly evaluated by light Microscope(10 x 100X) for Morphology, distribution of platelets, Giant platelets, platelet clumps by EDTA anticoagulant, platelet satellitism etc. and platelet count is also done examining 10 fields under oil immersion and multiplying the average platelet count of 10 fields by factor 15 as the method adopted by Ritu Bajpai, Chandra Rajak etc as cited in reference No. 5
  2. Platelet count is done by improved Neubauer counting chamber (which is more precise when the platelet count is less than 30000).
  3. Automated Hematological Analyzer (sysmax XNL 350, by impedance principal) all samples were evaluated by the automated analyzer and verified by peripheral blood smear examination and manual counting methods.

Other Methods:

  1. Optical method using two dimensions of light scattered such as incorporated in Bayer Adbia 120 and Abbot CD 4000. There is less chance of pseudo thrombocytopenia registered by the method.
  2. Immunological flow cytometric method: this is superior to optical method.

RESULTS: 

Blood samples of 1650 patients who reported to Malla Reddy Hospital Suraram, Hyderabad to Inpatient or Outpatient department with the age group of 4 days to 85 years, were collected in EDTA vaccutainer as per standard operating procedure and platelet count estimated by automated cell counter with a period from 27-5-2019 to 9-6-2019. 236 blood samples of the above patients with low platelet count (less than normal 1.5 lakhs) were registered.

All the 236 patients blood samples with low blood platelet counts were evaluated and verified by peripheral blood smear and manual counts usingImproved Neubauer counting chamber. It was observed that 71(30%) blood samples shows variations in platelet counts. 63 (27%) blood samples shows less platelet counts(ranging from 4000-1.06 lakhs) by the automated counter than actual count as verified by peripheral blood smear and manual counting method.

8 (3%) Blood samples show more platelet counts (6000-1.06 lakhs) by the automated counter than the actual platelet count as verified by peripheral blood smear and manual counting method. The results are summarized in Table no 1.

Age wise distribution and sex wise distribution of patients showing variation in platelet count by automated machine is shown in Tables No. 2 and 3 respectively.

DISCUSSION:

It is very important to evaluate accurate platelet count for management of Thrombocytopenia. Mostly automated counter provides accurate platelet counts but at times they are unable to discriminate platelets from non platelet particles like fragments of White blood cells, red blood cells, cellular debris, lipids and proteins aggregates, platelet abnormal distributions, large platelets, small platelets and platelets count less than 3000 which show false platelet count. In the present study 71 patients (30% cases) showed variation in platelet count out of which 63 patients (27% cases) showed lower platelet count than actual count and 8 patients (3% cases) showed lower platelet count than actual platelet count by automated counts with a variation of 4000 to 1.06 lakhs platelet counts as verified by the peripheral blood smear examination and Manual count.

In the study of Webb et al reviewed 35 samples with normal low and high platelet count. They compared the smear assessment with automated counter results. There was fair concordance in 27 specimens. In 3 specimens under estimations were found, over estimations in 5 specimens. 12

A cross section study conducted in National Centre for Public Health Laboratories at Aden Yemen by Bakhubaria, found that the mean platelet count estimated by manual method was not significantly different from the estimation by electronic method. 13

In another study conducted by Ritu Bajpai et al, it was observed that mean platelet count estimated by manual method for the sample studied has no significance (ᴘ =0.69) difference of value between manual method (0.94±0.29 lakhs/mm3) and the automated method (0.91± 0.27 lakhs/mm3). 5

The standard deviations of platelet count in the whole blood by automated and manual method are 28579.84 and 27163.94 respectively.

In the present study there is a platelet count deviation of 4000-1.6 lakh between the manual count and the automated count as the automated counter has not registered the non platelet particles, giant platelets and platelet aggregates. 

CONCLUSION:

It  is mandatoryto evaluate platelet countsobtained by automated machines, by peripheral smear examination and manual platelet count especially when the counts are less than 30 x 109/L

REFERENCES:

  1. Hartwing HJ. Platelet Morphology. In; Loscalzo J, Schafer IA, editors. Thrombosis and Hemorrhage. 2nd Ed, Baltimore, Williams & Wilkins; 1998, p. 207.
  2. Nurden AT, Nurden D, Sanchez M, Andre I, Anituo E. Platelets and wound healing. Front Biosci 2008;13:3532-48.
  3. Behnke O, Forer A. Blood platelets heterogeneity evidence for two classes of platelets in man and rat Br J Hematol 1993;94:686-93.
  4. Kunz D. Possibility and limitation of automated Platelet Counting procedure in the Thrombocytopenic range. Semin Thromb Hemost 2001; 27:229 – 235.
  5. Bajpai R, Rajak C, Poonia M. Platelet estimation by Peripheral Smear; Reliable, rapid, cost effective method to assess degree of thrombocytopenia. Int J Med Sci Res Pract 2015; 2(2):90 – 93.
  6. Hanseler E, Fehr J, Keller J. Estimation of lower limit of manual and automated platelet counting. Am J Clin Pathol 1997; 57: 629 – 636.
  7. Leeta JB, Mrudula, Devaraju S, Saldhana C, Veronica. A Comparative study of platelet count by manual and automated method in platelet poor plasma. Int J Recent Trends Sci Technol 2014;12(2):262-265.
  8. Dacie and Lewis practical hematology by Barbare J. Bain, Inceda Bales &others 11th Edition 2012;431-438.
  9. Onder O, Wernstein A. Pseudo thrombocytopenia caused by platelet agglutination that is reactive in blood anticoagulant and chelating agent. Blood. 1980;56:177.
  10. Gripper, Gratnick HR. Platelet to leucocytic adherence phenomena associated with thrombocytopenia Blood 1976; 22:460.
  11. Oliveria RAG, Marika M, Dachi T. Is automated platelet counting still a problem in thrombocytopenic blood? Sao Paulo, Med J/Rerv Paulo Med 2003;121(1):19-23.
  12. Webb DL, Parker L, Webb K. Platelet count assessment from peripheral blood smear. Alaska Med 2004;46:92-95.
  13. Bakhubaira S. Automated versus manual platelet count in Aden. J Clin Exp Pathol 2013;3:3

Figure 1: Normal platelet counts (Leishmans stain 10 x 100)

Figure 2: Platelets seen in clumps (Leishmans stain 10 x 100)

Figure 3: Giant Platelet (Leishmans stain 10 x 100)





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